Amylin analogues

ABSTRACT

The present invention relates to amylin analogues and to their use in the treatment or prevention of a variety of diseases, conditions or disorders, including obesity, excess food intake and associated metabolic diseases such as diabetes. The analogues have good physical and chemical stability, good solubility, and a long duration of action, and are well suited for use in the form of a liquid formulation.

PRIORITY

This application claims priority from European patent application no.16188024.0, filed 9 Sep. 2016, the disclosure of which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to amylin analogues that are amylinreceptor agonists, and to their medical use in the treatment and/orprevention of a variety of diseases, conditions or disorders, includingtreatment and/or prevention of excess food intake, obesity and excessbody weight, metabolic diseases, and other conditions and disordersdescribed herein. In particular, the present invention relates to stableamylin analogues that have a long duration of action and are well suitedfor use in the form of a liquid formulation.

BACKGROUND OF THE INVENTION

Amylin is one of a family of peptide hormones that includes amylin,calcitonin, calcitonin gene-related peptide, adrenomedullin andintermedin (intermedin also being known as AFP-6), and has beenimplicated in various metabolic diseases and disorders. Human amylin wasfirst isolated, purified and characterized as the major component ofamyloid deposits in the islets of pancreases from type 2 diabetespatients.

Native human amylin is a 37-amino acid peptide having the formula

H-KCONTATCOATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH₂

wherein H- at the N-terminus designates a hydrogen atom, correspondingto the presence of a free amino group on the N-terminal amino acidresidue [i.e. the lysine (K) residue at sequence position number 1 inthe sequence shown above]; wherein —NH₂ at the C-terminus indicates thatthe C-terminal carboxyl group is in the amide form; and wherein theparentheses ( ) associated with the two cysteine (C, Cys) residues atsequence positions 2 and 7 indicate the presence of an intramoleculardisulfide bridge between the two Cys residues in question.

Amylin may be beneficial in treating metabolic disorders such asdiabetes and/or obesity. Amylin is believed to regulate gastricemptying, and to suppress glucagon secretion and food intake, therebyregulating the rate of glucose release to the circulation. Amylinappears to complement the actions of insulin. Compared to healthyadults, type 1 diabetes patients have no circulating amylin, and type 2diabetes patients exhibit reduced postprandial amylin concentrations. Inhuman trials an amylin analogue known as pramlintide, described in WO93/10146 and having the sequenceLys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr, which also possesses a disulphide bridge betweenthe Cys residues at positions 2 and 7, has been shown to reduce bodyweight or reduce weight gain. An alternative amylin analogueincorporating N-methylated residues and having a reduced tendency tofibrillation, designated IAPP-GI, has been described by Yan et al.(PNAS, 103(7), 2046-2051, 2006; Angew. Chem. Int. Ed. 2013, 52,10378-10383; WO2006/042745). IAPP-GI appears to have lower activity thannative amylin, however.

WO91/07978 describes analogues of hypocalcemic peptides, includingamylin, in which internal disulphide bridges are replaced withalternative cyclisations. The effect of these alternative structures onthe activity of amylin analogues is not disclosed. WO99/34764 presentsdata showing that ^(2,7)cyclo-[2Asp,7Lys]-h-amylin has considerablylower potency than certain other amylin analogues, and human amylinitself.

Further analogues of amylin or pramlintide are described inWO2013/156594, WO2012/168430, WO2012/168431 and WO2012/168432, as wellas WO2015/040182.

Obesity is believed to be a major causal factor in development of type 2diabetes, which constitutes a growing and worldwide major healthproblem. Diseases or disorders that may develop as a consequence ofuntreated diabetes include cardiovascular and peripheral artery disease,micro- and macrovascular complications, stroke, and certain forms ofcancer, particularly hematopoietic cancers.

There is a need in the art for further amylin analogues. For example,amylin analogues that show a reduced tendency for fibrillation and/orhigh chemical stability at or around pH 7 might allow for a formulationat or near physiological pH. Amylin analogues having appropriately longplasma elimination half-lives, may also enable longer intervals betweendosing than is currently possible (e.g. once weekly, or even lessfrequently) and hence improve patient compliance. High levels of agonistactivity at the amylin receptor may also be desirable.

SUMMARY OF THE INVENTION

The present invention relates to compounds which are analogues of humanamylin.

In a first aspect, the invention provides an amylin analogue which is acompound having the formula:

R¹—Z—R²

wherein

R¹ is hydrogen, C₁₋₄ acyl, benzoyl or C₁₋₄ alkyl, or a half-lifeextending moiety M, wherein M is optionally linked to Z via a linkermoiety L;

R² is OH or NHR³, wherein R³ is hydrogen or C₁₋₃-alkyl; and

Z is an amino acid sequence of formula I:

(I) (SEQ ID NO: 3) X1-X2-X3-X4-X5-X6-X7-Ala-Thr-X10-Arg-Leu-Ala-X14-Phe-Leu-X17-Arg-X19-X20-Phe-Gly(Me)-Ala-lle(Me)-X27-Ser-Ser-Thr-Glu-X32-Gly-Ser-X35-Thr-X37

wherein

X1 is selected from the group consisting of Arg, Lys and Glu;

X3 is selected from the group consisting of Gly, Gln and Pro;

X4 is selected from the group consisting of Thr and Glu;

X5 is selected from the group consisting of Ala and Leu;

X6 is selected from the group consisting of Thr and Ser;

X10 is selected from the group consisting of Glu and Gln;

X14 is selected from the group consisting of Aad, His, Asp, Asn and Arg;

X17 is selected from the group consisting of Gln, His and Thr;

X19-X20 is selected from Ser-Ser, Thr-Thr, Ala-Thr, Ala-Ala, Gly-Thr,Gly-Gly and Ala-Asn or is absent;

X27 is selected from the group consisting of Leu and Pro;

X32 is selected from the group consisting of Val and Thr;

X35 is selected from the group consisting of Asn and Ser;

X37 is selected from the group consisting of Hyp and Pro; and

X2 and X7 are amino acid residues whose side chains together form alactam bridge;

or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, X1 is selected from Arg and Lys.

In some embodiments, X3 is Gly, X4 is Thr, X5 is Ala and/or X6 is Thr,e.g. X3 is Gly, X4 is Thr, X5 is Ala and X6 is Thr.

In some embodiments, X14 is selected from His, Asp and Aad.

In some embodiments, X17 is Gln.

In some embodiments, X19-X20 is selected from Ser-Ser and Thr-Thr, or isabsent, e.g. Ser-Ser.

In some embodiments, X32 is Val, X35 is Asn and/or X37 is Hyp.

Thus, Z may be an amino acid sequence of formula II:

(II) (SEQ ID NO: 4) X1-X2-Gly-Thr-Ala-Thr-X7-Ala-Thr-X10-Arg-Leu-Ala-X14-Phe-Leu-Gln-Arg-X19-X20-Phe-Gly(Me)-Ala-Ile(Me)-X27-Ser-Ser-Thr-Glu-Val-Gly-Ser-Asn-Thr-Hyp

wherein

X1 is selected from the group consisting of Arg and Lys;

X10 is selected from the group consisting of Glu and Gln;

X14 is selected from the group consisting of Aad, Asp and His;

X19-X20 is selected from Ser-Ser and Thr-Thr or is absent;

X27 is selected from the group consisting of Leu and Pro; and

X2 and X7 are amino acid residues whose side chains together form alactam bridge.

In some embodiments, X14 is Aad, X19-X20 is Ser-Ser and X27 is Leu.

Throughout this specification, amino acid positions of the amylinanalogues are numbered according to the corresponding position in nativehuman amylin having the sequence shown above. The sequence of Formulae Iand II (and other formulae herein) contain a two amino acid deletioncorresponding to the two residues Asn21 and Asn22 of human amylin. Thus,for ease of comparison with the amylin sequence, the Phe residueimmediately C-terminal (downstream) of position X20 is designated asposition 23, since it aligns with Phe23 of human amylin. Thus, thenumbering of any given residue in Formulae I and II above, and in otherformulae elsewhere in this specification, reflects the correspondingresidue in human amylin when optimally aligned therewith and does notnecessarily reflect its linear position in the particular sequence.

(It will be apparent that any of the relevant formulae presented in thisspecification could be written to include residues X21-X22 at theappropriate positions, wherein X21 and X22 are absent.)

Native amylin is known to form fibrils in aqueous solution almostinstantly. Consequently, many attempts have been made to enhance thestability of amylin analogues in liquid formulations. The tendency forfibrillation can be reduced by incorporating N-methylated residues (asmentioned above) and/or by substitution of certain amino acids atvarious positions. However, despite these options the desire to furtheroptimize the stability of amylin analogues in aqueous solution remains.Amylin analogues with further enhanced chemical stability in aqueoussolution would facilitate development of a corresponding pharmaceuticalproduct, potentially even in the form of a ready-to-use formulation,e.g. at or around the neutral pH range (pH 7-7.4).

Native amylin, and the vast majority of amylin analogues (such aspramlintide) contain a disulphide bridge between cysteine residues atpositions 2 and 7. The internal cyclisation which this bridge providesappears to be required for full potency and activity. Although compoundscontaining an internal disulphide bond are frequently less chemicallystable than might be desired, and the presence of the bond maycontribute to dimerisation and oligomerisation, e.g. via disulphideexchange reactions, the disulphide bond in amylin or amylin analogueshas not been reported to be a factor relevant for the low chemicalstability in aqueous formulations.

Few attempts to replace the disulphide bridge in amylin have beenreported. As described above, in an attempt to enhance in vivo stabilityand efficacy by reducing chemical and enzymatic proteolysis, WO91/07978proposes replacing internal disulphide bridges of hypocalcemic peptides(including e.g. calcitonin and amylin) with alternative cyclisations.However, the effect of these alternative structures on the activity ofamylin analogues is not disclosed. WO99/34764 presents data showing thatreplacement of the disulphide bridge with an intramolecular lactambridge in the native human amylin sequence results in an amylin analogue(^(2,7)cyclo-[2Asp,7Lys]-h-amylin) having considerably lower potencythan the wild type and many other amylin analogues, which may furtherexplain why alternative cyclisation options have not been pursuedfurther.

However, it has now been found that lactam-based cyclisations are verycompatible with amylin analogues having deletions at positions 21 and22, as the replacement of the disulphide bridge by a lactam bridge leadsto a substantial increase of stability in aqueous solution (see table 2)while other beneficial properties of these amylin analogues, such as lowtendency for fibrillation, high activity and good solubility areretained. Although it is known that such lactam bridges reduce theactivity of the peptides dramatically (see WO 91/07978, p 45 lines 36 to52; WO 99/34764 p. 84, Table A), in addition it has now been found thatamylin analogues of the present invention may retain high activity/showno reduction of activity at the hCT-R, hAMYR1, hAMYR2 and/or hAMYR3receptors. Additionally or alternatively they may have excellentchemical stability and resistance to fibrillation, especially but notexclusively in the neutral pH range.

Thus the amylin analogue of the invention comprises a lactam bridgeformed between the side chains of the residues at positions X2 and X7.For simplicity, positions 2 and 7 will be discussed by reference to theresidues nominally present before lactam formation.

One of the residues at positions X2 and X7 is a residue with a sidechain comprising a carboxylic acid group and the other is a residue witha side chain comprising an amine group, wherein a lactam (cyclic amide)is formed between the carboxylic acid and amine groups. The amine may bea primary or secondary amine, but is typically a primary amine. Suitableamino acids whose side chains can participate in a lactam bridge includeAsp, Glu and Aad (having side chains comprising carboxylic acid groups)and Dap, Dab, Orn, Lys and hLys (having side chains comprising aminegroups). Any of the amino acids selected from Asp, Glu and Aad may inprinciple form a lactam bridge with any of the amino acids selected fromthe group consisting of Dap, Dab, Orn, Lys and hLys.

Thus, one of the residues at position X2 and X7 may be selected fromAsp, Glu and Aad, and the other may be selected from Dap, Dab, Orn, Lysand hLys.

In some embodiments the carboxylic acid component of the lactam bridgederives from the amino acid at position X2, whereas the amine componentof the lactam bridge derives from the amino acid at position X7. Thus X2may be selected from Asp, Glu and Aad, and X7 may be selected from Dap,Dab, Orn, Lys and hLys.

It may be beneficial that the side chain of the residue at position X2is of the same length as, or shorter than, the side chain of the residueat position X7. Such residue combinations can provide benefits includinghigher potency as compared to other combinations.

In this context, side chain length is counted as the number of atoms ina linear chain from the first atom of the side chain (which is bonded toan atom of the peptide backbone, i.e. to the alpha carbon of therelevant residue for most amino acids) up to and including the atomwhich participates in the amide bond of the lactam bridge (i.e. thecarbon atom of the carboxylic acid functional group or the nitrogen atomof the amine group).

Thus common acid- and amine-containing side chains are considered tohave the following side chain lengths:

Asp: 2 atoms

Glu: 3 atoms

Aad: 4 atoms

Dap: 2 atoms

Dab: 3 atoms

Orn: 4 atoms

Lys: 5 atoms

hLys: 6 atoms

In some embodiments the side chain of the residue at position X2 isshorter than the side chain of the residue at position X7.

Desirably, the length of the lactam bridge provided by the two sidechains after formation of the amide bond (not including any atoms in thepeptide backbone) is 4, 5, 6, 7 or 8 atoms, e.g. 5, 6, 7 or 8 atoms, or5, 6 or 7 atoms.

Thus, suitable pairings of residues at positions X2 and X7 in which theside chain at position X2 is shorter than the side chain at position X7include:

X2 is Asp and X7 is Lys

X2 is Asp and X7 is Orn

X2 is Asp and X7 is Dab

X2 is Asp and X7 is hLys

X2 is Dap and X7 is Aad

Examples of suitable pairings having the same side chain lengthsinclude:

X2 is Glu and X7 is Dab

X2 is Dab and X7 is Glu

Further pairings which may nevertheless be considered include:

X2 is Asp and X7 is Dap

X2 is Aad and X7 is Dap

X2 is Dap and X7 is Asp

X2 is Dab and X7 is Asp

X2 is Orn and X7 is Asp

Pairings of particular interest are:

X2 is Asp and X7 is Lys

X2 is Asp and X7 is Orn.

In some embodiments of the formulae described above:

X1 may be Arg;

X10 may be Glu.

X14 may be selected from Asp and Aad.

X19-X20 may be Ser-Ser

X27 may be Leu

The amylin analogue may have the formula:

R¹—Z—R²

wherein

R¹ is hydrogen, C₁₋₄ acyl, benzoyl or C₁₋₄ alkyl, or a half-lifeextending moiety M, wherein M is optionally linked to Z via a linkermoiety L;

R² is OH or NHR³, wherein R³ is hydrogen or C₁₋₃-alkyl; and

Z is an amino acid sequence selected from the group consisting of:

(SEQ ID NO: 5)RD()GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 6)RD()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 7)RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 8)RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 9)RD()GTAT-Orn()-ATERLAHFLQRF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 10)RD()GTAT-Orn()-ATERLAHFLHRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNTP(SEQ ID NO: 11)RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 12)RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 13)RD()GTAT-Orn()-ATERLARFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 14)ED()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 15)RD()GEATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 16)RD()GTLTK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 17)RD()GTASK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 18)RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 19)RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 20)RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 21)RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 22)RD()GTAT-hLys()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 23)RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 24)RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 25)RD()GTAT-Orn()-ATERLA-Aad-FLTRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSST-Hyp(SEQ ID NO: 26)RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 27)RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 28)RD()GTAT-Orn()-ATERLA-Aad-FLQRATF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 29)RD()GTAT-Orn()-ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 30)RD()GTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 31)RD()QTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 32)RD()PTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 33)ED()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 34)RD()GTATK()ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 35)RD()GTATK()ATERLA-Aad-FLQRGGF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 36)RD()GTATK()ATERLA-Aad-FLQRANF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 37)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 38)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 39)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 40)ED()GTATK()ATERLA-Aad-FLQRSSFGly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 41)RD()GTATK()ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 42)KD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-AIle(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 43)RD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 44)RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 45)RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 46)KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 47)KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 48)R-Dap()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 49)R-Dab()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 50)R-Orn()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 51)R-Dap()-GTAT-Aad()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 52)R-Dab()-GTATE()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 53)R-Aad()-GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 54)RE()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp

or a pharmaceutically acceptable salt or solvate thereof.

The lactam bridge at positions 2 and 7 is indicated by parentheses ( )following the residues at those positions.

In some embodiments, R¹ is M or M-L-, and/or R² is NH₂.

Specific compounds of the invention include:

(Compound 1) (SEQ ID NO: 55)[19CD]-isoGlu-RD()GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 2) (SEQ ID NO: 56)[19CD]-isoGlu-RD()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 3) (SEQ ID NO: 57)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 4) (SEQ ID NO: 58)[19CD]-isoGlu-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 5) (SEQ ID NO: 59)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 6) (SEQ ID NO: 60)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 7) (SEQ ID NO: 61)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLHRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNTP-NH₂ (Compound 8) (SEQ ID NO: 62)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 9) (SEQ ID NO: 63)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 10) (SEQ ID NO: 64)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLARFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 11) (SEQ ID NO: 65)[19CD]-isoGlu-ED()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 12) (SEQ ID NO: 66)[19CD]-isoGlu-RD()GEATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 13) (SEQ ID NO: 67)[19CD]-isoGlu-RD()GTLTK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 14) (SEQ ID NO: 68)[19CD]-isoGlu-RD()GTASK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 15) (SEQ ID NO: 69)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 16) (SEQ ID NO: 70)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 17) (SEQ ID NO: 71)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 18) (SEQ ID NO: 72)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 19) (SEQ ID NO: 73)[19CD]-isoGlu-RD()GTAT-hLys()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 20) (SEQ ID NO: 74)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 21) (SEQ ID NO: 75)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 22) (SEQ ID NO: 76)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLTRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSST-Hyp-NH₂ (Compound 23) (SEQ ID NO: 77)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 24) (SEQ ID NO: 78)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 25) (SEQ ID NO: 79)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRATF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 26) (SEQ ID NO: 80)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 27) (SEQ ID NO: 81)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 28) (SEQ ID NO: 82)[19CD]-isoGlu-RD()QTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 29) (SEQ ID NO: 83)[19CD]-isoGlu-RD()PTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 30) (SEQ ID NO: 84)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 31) (SEQ ID NO: 85)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 32) (SEQ ID NO: 86)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRGGF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 33) (SEQ ID NO: 87)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRANF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 34) (SEQ ID NO: 88)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 35) (SEQ ID NO: 89)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 36) (SEQ ID NO: 90)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 37) (SEQ ID NO: 91)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSFGly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 38) (SEQ ID NO: 92)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLORTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 39) (SEQ ID NO: 93)[19CD]-isoGlu-KD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-AIle(Me)-LSSTEVGSNTHyp-NH₂ (Compound 40) (SEQ ID NO: 94)[19CD]-isoGlu-RD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 41) (SEQ ID NO: 95)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 42) (SEQ ID NO: 96)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 43) (SEQ ID NO: 97)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 44) (SEQ ID NO: 98)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 45) (SEQ ID NO: 99)[19CD]-isoGlu-R-Dap()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 46) (SEQ ID NO: 100)[19CD]-isoGlu-R-Dab()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 47) (SEQ ID NO: 101)[19CD]-isoGlu-R-Orn()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 48) (SEQ ID NO: 102)[19CD]-isoGlu-R-Dap()-GTAT-Aad()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 49) (SEQ ID NO: 103)[19CD]-isoGlu-R-Dab()-GTATE()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 50) (SEQ ID NO: 104)[19CD]-isoGlu-R-Aad()-GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 51) (SEQ ID NO: 105)[19CD]-isoGlu-RE()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂

and pharmaceutically acceptable salts and solvates thereof.

The invention further provides a composition comprising an amylinanalogue as described above. The composition may be a pharmaceuticalcomposition, and may comprise a pharmaceutically acceptable carrier,excipient or vehicle.

The invention further provides a method for the synthesis of an amylinanalogue as described above. The method may comprise the steps ofsynthesising the peptide by solid-phase or liquid-phase methodology, andoptionally isolating and/or purifying the final product. The method mayfurther comprise the step of forming an amide bond between the sidechains at positions 2 and 7.

The present invention further provides an amylin analogue of theinvention for use in a method of medical treatment.

The amylin analogues are useful, inter alia, in the reduction of foodintake, promotion of weight loss, and inhibition or reduction of weightgain. As a result, they may be used for treatment of a variety ofconditions, diseases, or disorders in a subject, including, but notlimited to, obesity and various obesity-related conditions, diseases, ordisorders, such as diabetes (e.g. type 2 diabetes), hypertension,dyslipidemia, sleep apnea and cardiovascular disease. The subject may beaffected by obesity accompanied by at least one weight-related co-morbidcondition, such as diabetes (e.g. type 2 diabetes), hypertension,dyslipidemia, sleep apnea and cardiovascular disease. It will beunderstood that the amylin analogues may thus be administered tosubjects affected by conditions characterised by inadequate control ofappetite or otherwise over-feeding, such as binge-eating disorder andPrader-Willi syndrome. It will be clear that the analogues can be usedfor treatment of combinations of the conditions described.

Thus, the invention provides an amylin analogue of the invention for usein a method of treating, inhibiting or reducing weight gain, promotingweight loss and/or reducing excess body weight. Treatment may beachieved, for example, by control of appetite, feeding, food intake,calorie intake and/or energy expenditure.

The invention also provides an amylin analogue of the invention for usein a method of treating obesity as well as associated diseases,disorders and health conditions, including, but not limited to, morbidobesity, obesity prior to surgery, obesity-linked inflammation,obesity-linked gallbladder disease and obesity-induced sleep apnea andrespiratory problems, degeneration of cartilage, osteoarthritis, andreproductive health complications of obesity or overweight such asinfertility. The subject may be affected by obesity accompanied by atleast one weight-related co-morbid condition, such as diabetes (e.g.type 2 diabetes), hypertension, dyslipidemia, sleep apnea andcardiovascular disease.

The invention also provides an amylin analogue of the invention for usein a method of prevention or treatment of Alzheimer's disease, diabetes,type 1 diabetes, type 2 diabetes, pre-diabetes, insulin resistancesyndrome, impaired glucose tolerance (IGT), disease states associatedwith elevated blood glucose levels, metabolic disease includingmetabolic syndrome, hyperglycemia, hypertension, atherogenicdyslipidemia, hepatic steatosis (“fatty liver”; including non-alcoholicfatty liver disease (NAFLD), which itself includes non-alcoholicsteatohepatitis (NASH)), kidney failure, arteriosclerosis (e.g.atherosclerosis), macrovascular disease, microvascular disease, diabeticheart disease (including diabetic cardiomyopathy and heart failure as adiabetic complication), coronary heart disease, peripheral arterydisease or stroke, and combinations thereof.

The invention also provides an amylin analogue of the invention for usein a method of lowering circulating LDL levels and/or increasing HDL/LDLratio.

Effects of amylin analogues on these conditions may be mediated in wholeor in part via an effect on body weight, or may be independent thereof.

The invention further provides use of an amylin analogue of theinvention in the manufacture of a medicament for treating, inhibiting orreducing weight gain, promoting weight loss and/or reducing excess bodyweight.

The invention also provides use of an amylin analogue of the inventionin the manufacture of a medicament for treating obesity as well asassociated diseases, disorders and health conditions, including, but notlimited to, morbid obesity, obesity prior to surgery, obesity-linkedinflammation, obesity-linked gallbladder disease and obesity-inducedsleep apnea and respiratory problems, degeneration of cartilage,osteoarthritis, and reproductive health complications of obesity oroverweight such as infertility. The subject may be affected by obesityaccompanied by at least one weight-related co-morbid condition, such asdiabetes (e.g. type 2 diabetes), hypertension, dyslipidemia, sleep apneaand cardiovascular disease.

The invention also provides use of an amylin analogue of the inventionin the manufacture of a medicament for the prevention or treatment ofAlzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes,pre-diabetes, insulin resistance syndrome, impaired glucose tolerance(IGT), disease states associated with elevated blood glucose levels,metabolic disease including metabolic syndrome, hyperglycemia,hypertension, atherogenic dyslipidemia, hepatic steatosis (“fattyliver”; including non-alcoholic fatty liver disease (NAFLD), whichitself includes non-alcoholic steatohepatitis (NASH)), kidney failure,arteriosclerosis (e.g. atherosclerosis), macrovascular disease,microvascular disease, diabetic heart disease (including diabeticcardiomyopathy and heart failure as a diabetic complication), coronaryheart disease, peripheral artery disease or stroke, and combinationsthereof.

The invention also provides use of an amylin analogue of the inventionin the manufacture of a medicament for lowering circulating LDL levelsand/or increasing HDL/LDL ratio.

The invention further provides a method of treating, inhibiting orreducing weight gain, promoting weight loss and/or reducing excess bodyweight in a subject, comprising administering a therapeuticallyeffective amount of an amylin analogue of the invention to the subject.

The invention also provides a method of treating obesity as well asassociated diseases, disorders and health conditions, including, but notlimited to, morbid obesity, obesity prior to surgery, obesity-linkedinflammation, obesity-linked gallbladder disease and obesity-inducedsleep apnea and respiratory problems, degeneration of cartilage,osteoarthritis, and reproductive health complications of obesity oroverweight such as infertility in a subject, comprising administering atherapeutically effective amount of an amylin analogue of the inventionto the subject. The subject may be affected by obesity accompanied by atleast one weight-related co-morbid condition, such as diabetes (e.g.type 2 diabetes), hypertension, dyslipidemia, sleep apnea andcardiovascular disease.

The invention also provides a method of prevention or treatment ofAlzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes,pre-diabetes, insulin resistance syndrome, impaired glucose tolerance(IGT), disease states associated with elevated blood glucose levels,metabolic disease including metabolic syndrome, hyperglycemia,hypertension, atherogenic dyslipidemia, hepatic steatosis (“fattyliver”; including non-alcoholic fatty liver disease (NAFLD), whichitself includes non-alcoholic steatohepatitis (NASH)), kidney failure,arteriosclerosis (e.g. atherosclerosis), macrovascular disease,microvascular disease, diabetic heart disease (including diabeticcardiomyopathy and heart failure as a diabetic complication), coronaryheart disease, peripheral artery disease or stroke, and combinationsthereof, in a subject, comprising administering a therapeuticallyeffective amount of an amylin analogue of the invention to the subject.

The invention further provides a method of lowering circulating LDLlevels and/or increasing HDL/LDL ratio in a subject, comprisingadministering a therapeutically effective amount of an amylin analogueof the invention to the subject.

The invention further provides the use of an amylin analogue asdescribed above in a method of cosmetic (i.e. non-therapeutic) weightloss. It will be understood that references to therapeutic uses ofamylin analogues and methods comprising administration of amylinanalogues may equally be taken to encompass uses and administration ofsuch compositions.

Further aspects and embodiments of the present invention will becomeapparent from the disclosure below.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms usedherein shall have the meanings that are commonly understood by those ofordinary skill in the art. Generally, nomenclature employed herein inconnection with techniques of chemistry, molecular biology, cell andcancer biology, immunology, microbiology, pharmacology, and protein andnucleic acid chemistry, described herein, is that well known andcommonly used in the art.

All publications, patents and published patent applications referred toin this application are specifically incorporated by reference herein.In case of conflict, the present specification, including its specificdefinitions, will control.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or component, or of a stated group of integers orcomponents, but not the exclusion of any other integer or component orgroup of integers or components.

The singular forms “a,” “an,” and “the” include the plurals unless thecontext clearly dictates otherwise.

The term “including” is used to mean “including but not limited to.”“Including” and “including but not limited to” are used interchangeably.

The terms “patient”, “subject,” and “individual” may be usedinterchangeably and may refer to either a human or a non-human animal.Subjects are typically mammals, including humans, non-human primates(including great apes, Old World monkeys and New World monkeys),livestock animals (e.g., bovines, porcines), companion animals (e.g.,canines, felines) and rodents (e.g., mice and rats).

As used herein, the term “pharmaceutically acceptable salt” is intendedto indicate a salt which is not harmful to a patient or subject to whichthe salt in question is administered. It may suitably be a salt chosen,e.g., among acid addition salts and basic salts. Examples of acidaddition salts include chloride salts, citrate salts and acetate salts.Examples of basic salts include salts where the cation is selected amongalkali metal cations, such as sodium or potassium ions, alkaline earthmetal cations, such as calcium or magnesium ions, as well as substitutedammonium ions, such as ions of the type N(R¹)(R²)(R³)(R⁴)⁺, where R¹,R², R³ and R⁴ independently will typically designate hydrogen,optionally substituted C₁₋₆-alkyl or optionally substitutedC₂₋₆-alkenyl. Examples of relevant C₁₋₆-alkyl groups include methyl,ethyl, 1-propyl and 2-propyl groups. Examples of C₂₋₆-alkenyl groups ofpossible relevance include ethenyl, 1-propenyl and 2-propenyl. Otherexamples of pharmaceutically acceptable salts are described in“Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro(Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recenteditions thereof), in the “Encyclopaedia of Pharmaceutical Technology”,3^(rd) edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.),NY, USA, 2007, and in J. Pharm. Sci. 66:2 (1977).

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, apeptide or pharmaceutically acceptable salt thereof according to theinvention) and a solvent. The solvent in this connection may, forexample, be water, ethanol or another pharmaceuticallyacceptable—typically small-molecular—organic species, such as, but notlimited to, acetic acid or lactic acid. When the solvent in question iswater, such a solvate is normally referred to as a hydrate.

The term “agonist” as employed in the context of the invention refers toa substance that activates the receptor type in question, typically bybinding to it (i.e. as a ligand).

Each embodiment of the invention described herein may be taken alone orin combination with one or more other embodiments of the invention.

Throughout the present specification, unless naturally occurring aminoacids are referred to by their full name (e.g. alanine, arginine, etc.),they are designated by their conventional three-letter or single-letterabbreviations (e.g. Ala or A for alanine, Arg or R for arginine, etc.).In the case of certain less common or non-naturally occurring aminoacids (i.e. amino acids other than the 20 encoded by the standardmammalian genetic code), unless they are referred to by their full name(e.g. sarcosine, ornithine, etc.), frequently employed three- orfour-character codes are employed for residues thereof, including Orn(ornithine, i.e. 2,5-diaminopentanoic acid), Aib (α-aminoisobutyricacid), Dab (2,4-diaminobutanoic acid), Dap (2,3-diaminopropanoic acid),Har (homoarginine), γ-Glu (γ-glutamic acid), Gaba (γ-aminobutanoicacid), β-Ala (i.e. 3-aminopropanoic acid), 8Ado(8-amino-3,6-dioxaoctanoic acid).

Unless otherwise indicated, reference is made to the L-isomeric forms ofthe amino acids in question.

Additional abbreviations include the following:

-   -   Gly(Me): N-methylglycine [also known as sarcosine (Sar)]    -   Ile(Me): N-methylisoleucine    -   Aad: 2-aminoadipic acid, e.g. (2S)-2-aminoadipic acid [also        (2S)-2-aminohexanedioic acid], also known as homo-glutamic acid    -   Hyp: 4-hydroxyproline, e.g. (2S,4R)-4-hydroxyproline [also        denoted (4R)-4-hydroxy-L-proline]    -   Dap: 2,3-diaminopropanoic acid, e.g. (2S)-2,3-diaminopropanoic        acid    -   Dab: 2,4-diaminobutanoic acid, e.g. (2S)-2,4-diaminobutanoic        acid    -   hLys: 2-amino-7-amino-heptanoic acid, also known as homo-lysine,        e.g. (2S)-2-amino-7-amino-heptanoic acid

The term “therapeutically effective amount” as used herein in thecontext of the above-described methods of treatment or other therapeuticinterventions according to the invention refers to an amount that issufficient to cure, ameliorate, alleviate or partially arrest theclinical manifestations of the particular disease, disorder or conditionthat is the object of the treatment or other therapeutic intervention inquestion e.g. as measured by established clinical endpoints or otherbiomarkers (established or experimental). A therapeutically relevantamount may be determined empirically by one skilled in the art based onthe indication being treated or prevented and the subject to whom thetherapeutically relevant amount is being administered. For example, theskilled worker may measure one or more of the clinically relevantindicators of bioactivity described herein, e.g. plasma lipid levels,blood glucose levels or insulin release. The skilled worker maydetermine a clinically relevant amount through in vitro or in vivomeasurements. Other exemplary measures include weight gain, weight loss,and change in blood pressure.

An amount adequate to accomplish any or all of these effects is definedas a therapeutically effective amount. The administered amount and themethod of administration can be tailored to achieve optimal efficacy. Anamount effective for a given purpose will depend, inter alia, on theseverity of the disease, disorder or condition that is the object of theparticular treatment or other therapeutic intervention, on the bodyweight and general condition of the subject in question, on diet, onpossible concurrent medication, and on other factors well known to thoseskilled in the medical arts.

Determination of an appropriate dosage size and dosing regimen mostappropriate for administration of a peptide or pharmaceuticallyacceptable salt or solvate thereof according to the invention to a humanmay be guided by the results obtained by the present invention, and maybe confirmed in properly designed clinical trials. An effective dosageand treatment protocol may be determined by conventional means, startingwith a low dose in laboratory animals and then increasing the dosagewhile monitoring the effects, and systematically varying the dosageregimen as well. Numerous factors may be taken into consideration by aclinician when determining an optimal dosage for a given subject. Suchconsiderations are well known to the skilled person.

The terms “treatment” and grammatical variants thereof (e.g. “treated”,“treating”, “treat”) as employed in the present context refer to anapproach for obtaining beneficial or desired clinical results. For thepurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilization (i.e. not worsening) of state ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterm “treatment” includes inhibition or reduction of an increase inseverity of a pathological state or symptoms (e.g. weight gain orhyperglycemia) relative to the absence of treatment, and is notnecessarily meant to imply complete cessation of the relevant disease,disorder or condition.

The terms “prevention” and grammatical variants thereof (e.g.,“prevented”, “preventing”, “prevent”) as employed in the present contextrefer to an approach for hindering or preventing the development of, oraltering the pathology of, a condition, disease or disorder.Accordingly, “prevention” may refer to prophylactic or preventivemeasures. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, prevention or slowingof symptoms, progression or development of a disease, whether detectableor undetectable. A subject (e.g. a human) in need of “prevention” maythus be a subject not yet afflicted with the disease or disorder inquestion. The term “prevention” thus includes inhibiting or slowing theonset of disease relative to the absence of treatment, and is notnecessarily meant to imply permanent prevention of the relevant disease,disorder or condition.

Synthesis of Amylin Analogues

The invention further provides a method of synthesis of an amylinanalogue of the invention. The amylin analogues (which may also bereferred to as compounds or peptides) may suitably be manufactured bystandard synthetic methods. Thus, the peptides may be synthesized by,e.g., methods comprising synthesizing the peptide by standardsolid-phase or liquid-phase methodology, either stepwise or by fragmentassembly, and optionally isolating and purifying the final peptideproduct. In this context, reference may be made to WO 98/11125 or, interalia, Fields, G. B. et al., “Principles and Practice of Solid-PhasePeptide Synthesis”; in: Synthetic Peptides, Gregory A. Grant (ed.),Oxford University Press (2^(nd) edition, 2002) and the synthesisexamples herein. The method typically further comprises the step offorming an amide bond between the side chains at positions 2 and 7, e.g.as described below. In the case of solid phase synthesis, cyclisationmay be performed in situ on the solid phase (e.g. resin), i.e. beforeremoval of the peptide from the solid phase.

C₁₋₄ Acyl Groups

C₁₋₄ acyl groups that may be present as a group R¹ in the context ofcompounds of the present invention include formyl (i.e. methanoyl),acetyl (i.e. ethanoyl), propanoyl, 1-butanoyl and 2-methylpropanoylgroups.

C₁₋₄ Alkyl Groups

C₁₋₄ alkyl groups that may be present as a group R¹ in the context ofcompounds of the present invention include, but are not limited to, C₁₋₃alkyl groups, such as methyl, ethyl, 1-propyl or 2-propyl.

C₁₋₃ Alkyl Groups

C₁₋₃ alkyl groups that may be present as a group R³ in the context ofcompounds of the present invention include methyl, ethyl, 1-propyl and2-propyl.

Half-Life Extending Moieties M

As described herein, the N-terminal moiety R¹ in a compound of theinvention may be a half-life extending moiety M (sometimes referred toin the literature as, inter alia, a duration enhancing moiety or albuminbinding moiety), optionally linked (covalently attached) to the peptidemoiety Z via a linker moiety L. Among suitable half-life extendingmoieties are certain types of lipophilic substituents. Without wishingto be bound by any particular theory, it is thought that such lipophilicsubstituents (and other classes of half-life extending moieties) bindalbumin in the blood stream, thereby shielding the compound of theinvention from renal filtration as well as enzymatic degradation andthus possibly enhancing the half-life of the compound in vivo. Thelipophilic substituent may also modulate the potency of the compound asan agonist to the amylin (calcitonin) receptor.

The lipophilic substituent may be attached to the N-terminal amino acidresidue or to the linker L via an ester, a sulfonyl ester, a thioester,an amide, an amine or a sulfonamide. Accordingly it will be understoodthat preferably the lipophilic substituent includes an acyl group, asulfonyl group, an N atom, an O atom or an S atom which forms part ofthe ester, sulfonyl ester, thioester, amide, amine or sulfonamide.Preferably, an acyl group in the lipophilic substituent forms part of anamide or ester with the amino acid residue or the linker.

The lipophilic substituent may comprise a hydrocarbon chain having from10 to 24 C atoms, e.g. from 14 to 22 C atoms, e.g. from 16 to 20 Catoms. Preferably it has at least 14 C atoms, and preferably has 20 Catoms or fewer. For example, the hydrocarbon chain may contain 14, 15,16, 17, 18, 19 or 20 carbon atoms. The hydrocarbon chain may be linearor branched, and may be saturated or unsaturated. Furthermore, it caninclude a functional group at the end of the hydrocarbon chain, e.g. acarboxylic acid group which may or may not be protected duringsynthesis. From the discussion above it will also be understood that thehydrocarbon chain is preferably substituted with a moiety which formspart of the attachment to the N-terminal amino acid residue of thepeptide moiety Z or to the linker L, for example an acyl group, asulfonyl group, an N atom, an O atom or an S atom.

Most preferably, the hydrocarbon chain is substituted with an acylgroup, and accordingly the hydrocarbon chain may be part of an alkanoylgroup, for example a dodecanoyl, 2-butyloctanoyl, tetradecanoyl,hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl or eicosanoylgroup. Examples of functionalized hydrocarbon chains are15-carboxy-pentadecanoyl, 17-carboxy-heptadecanoyl and19-carboxy-nonadecanoyl.

As mentioned above, a lipophilic substituent M may be linked to theN-terminal amino acid residue of Z via a linker L. In embodiments, thelinker moiety L may itself comprise one, two, three or more linkedsub-moieties L¹, L², L³, . . . etc. When the linker L comprises only onesuch moiety, it is attached to the lipophilic substituent and to theN-terminal amino acid residue of Z. The linker may then be attached tothe lipophilic substituent and to the N-terminal amino acid residue of Zindependently by means of an ester, a sulfonyl ester, a thioester, anamide, an amine or a sulfonamide bond. Accordingly, it may include twomoieties independently selected from acyl, sulfonyl, an N atom, an Oatom and an S atom. The linker may consist of a linear or branched C₁₋₁₀hydrocarbon chain or more preferably a linear C₁₋₅ hydrocarbon chain.Furthermore the linker can be substituted with one or more substituentsselected from C₁₋₆ alkyl, amino C₁₋₆ alkyl, hydroxy C₁₋₆ alkyl andcarboxy C₁₋₆ alkyl.

In some embodiments the linker may comprise one or more (e.g. one, twoor three) linked amino acid residues, which may each independently be aresidue of any naturally occurring or non-naturally occurring aminoacid. For example, the linker may comprise one, two or three linkedamino acid residues, each of which may independently be a residue ofGly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg,Gln, Asn, α-Glu, γ-Glu, ε-Lys, Asp, β-Asp, Ser, Thr, Gaba, Aib, β-Ala(i.e. 3-aminopropanoyl), 4-aminobutanoyl, 5-aminopentanoyl,6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl,10-aminodecanoyl or 8Ado (i.e. 8-amino-3,6-dioxaoctanoyl).

References to γ-Glu, ε-Lys, and β-Asp indicate residues of amino acidswhich participate in bonds via their side chain carboxyl or aminefunctional groups. Thus γ-Glu, and β-Asp participate in bonds via theiralpha amino and side chain carboxyl groups, while ε-Lys participates viaits carboxyl and side chain amino groups. In the context of the presentinvention, γ-Glu and isoGlu are used interchangeably.

In certain embodiments, the linker comprises or consists of one, two orthree independently selected residues of Glu, γ-Glu, ε-Lys, β-Ala,4-aminobutanoyl, 8-aminooctanoyl or 8Ado.

Linkers consisting of isoGlu and isoGlu-isoGlu may be particularlypreferred.

An example of a lipophilic substituent comprising a lipophilic moiety Mand linker L is shown in the formula below:

Here, the backbone nitrogen of an Arg residue (present at position X1 ofthe amylin analogue's peptide sequence Z) is covalently attached to theside chain carboxyl group of a Glu moiety via an amide linkage. A19-carboxy-nonadecanoyl group is covalently attached to the alpha aminogroup of the Glu linker via an amide linkage. Thus the Glu linker is inan iso-Glu (or γ-Glu) configuration. This combination of lipophilicmoiety and linker, attached to an Arg residue, may be referred to by theshorthand notation [19CD]-isoGlu-R, e.g. when shown in formulae ofspecific compounds.

The skilled person will be well aware of suitable techniques forpreparing the compounds employed in the context of the invention. Forexamples of suitable chemistry, see, e.g., WO98/08871, WO00/55184,WO00/55119, Madsen et al (J. Med. Chem. 2007, 50, 6126-32), and Knudsenet al. 2000 (J. Med Chem. 43, 1664-1669).

The hydrocarbon chain in a lipophilic substituent may be furthersubstituted. For example, it may be further substituted with up to threesubstituents selected from NH₂, OH and COOH. If the hydrocarbon chain isfurther substituted, it is preferably further substituted with only onesubstituent. Alternatively or additionally, the hydrocarbon chain mayinclude a cycloalkane or heterocycloalkane moiety, for example as shownbelow:

In some embodiments, the cycloalkane or heterocycloalkane moiety is asix-membered ring, e.g. a piperidine ring.

In alternative embodiments of the present invention, the N-terminalamino acid of Z in a compound of the invention may be linked (covalentlyattached) to a biotinylic substituent, optionally via a linker moiety L.Without wishing to be bound by any particular theory, it is likewisebelieved that such biotinylic substituents bind to albumin in the bloodstream, thereby shielding the compound of the invention from enzymaticdegradation and thus possibly enhancing the half-life of the compound invivo. A linker, when present, may provide spacing between the peptidemoiety Z and the biotinylic substituent.

The biotinylic substituent may be attached to the N-terminal amino acidresidue or to the linker via an maleimide ester bond, a sulfonyl esterbond, a thioester bond, an amide bond, an amine bond or a sulfonamidebond. Accordingly it will be understood that the biotinylic substituentpreferably comprises an maleimido group, an acyl group, a sulfonylgroup, an N atom, an O atom or an S atom which forms part of the ester,sulfonyl ester, thioester, amide, amine or sulfonamide bond in question.

Examples of biotinylic substituents may include

Biotin is known as Vitamin H or Coenzyme R, and is a water-solubleB-complex vitamin (vitamin B7). It has been shown to increase oraluptake of certain drugs.

Efficacy of Compounds

The compounds of the invention are amylin receptor agonists, i.e. theyare capable of binding to, and inducing signaling by, one or morereceptors or receptor complexes regarded as physiological receptors forhuman amylin. These include the human calcitonin receptor hCT-R, as wellas complexes comprising the human calcitonin receptor hCT-R and at leastone of the human receptor activity modifying proteins designated hRAMP1,hRAMP2 and hRAMP3. Complexes between hCT-R and hRAMP1, hRAMP2 and hRAMP3are designated hAMYR1, hAMYR2 and hAMYR3 (i.e. human amylin receptors 1,2 and 3) respectively.

Without wishing to be bound by theory, a compound may be considered anamylin receptor agonist if it has agonist activity at one or more ofhAMYR1, hAMYR2 and hAMYR3, e.g. against hAMYR1 and/or hAMYR3, e.g. athAMYR3.

Typically an amylin receptor agonist will also have agonist activity athCT-R when expressed in the absence of hRAMP1, hRAMP2 and hRAMP3.Typically, the agonist will have activity at hCT-R (when expressed inthe absence of hRAMP1, hRAMP2 and hRAMP3) which is less than 10-foldhigher than its activity at any one of hAMYR1, hAMYR2 and hAMYR3 (i.e.its activity at all of these receptors) in a comparable assay. Agonistactivity at hCT-R may be less than 5-fold higher than agonist activityat hAMYR1, hAMYR2 and hAMYR3, substantially equal to (e.g. +/−10%)agonist activity at hAMYR1, hAMYR2 and hAMYR3, or less than agonistactivity at hAMYR1, hAMYR2 and hAMYR3. In this regard, it may besufficient just to compare activity between hCT-R and hAMYR3.

The ability to induce cAMP formation (i.e. to induce adenylate cyclaseactivity) as a result of binding to the relevant receptor or receptorcomplex is typically regarded as indicative of agonist activity. Otherintracellular signaling pathways or events may also be used as read-outsfor amylin receptor agonist activity. These may include calcium release,β-arrestin recruitment, receptor internalization, kinase activation orinactivation, lipase activation, inositol phosphate release,diacylglycerol release or nuclear transcription factor translocation.

A suitable comparable assay format would utilize cells which expresshCT-R and which differ only in their expression of hRAMP1, 2 and 3. Forexample, a “base” cell line which does not express any of hCT-R, hRAMP1,hRAMP2 and hRAMP3 may be engineered to generate cells which express (i)hCT-R, and (ii) one of hAMYR1, hAMYR2 and hAMYR3 (i.e. hCT-R plus one ofhRAMP1, hRAMP2 and hRAMP3), e.g. hAMYR3. The base cells will typicallybe mammalian cells and may be primate cells. They may be non-humanprimate cells. Preferably the base cell does not express any of CT-R,RAMP1, RAMP2 or RAMPS (whether human, or native to the base cell if thebase cell is non-human). The base cells may be fibroblast cells.Suitable non-human fibroblast base cells include COS7 cells, fromAfrican green monkey, which do not express native CT-R or RAMPs.

Comparative activity may be measured by any suitable means, such as viadetermination of EC₅₀ values as described below. It will be apparentthat the same biological read-out must be for both receptor types.

Compounds of the present invention may exhibit a number of advantageousproperties in relation to human amylin and existing analogues thereof,such as pramlintide, IAPP-GI, and analogues described in WO2012/168430,WO2012/168431 and WO2012/168432. As compared to human amylin or any ofthose analogues, compounds of the invention may, for example, exhibitimproved efficacy (e.g., in the form of improved in vitro activity orpotency at one or more of the receptors hCT-R, hAMYR1, hAMYR2 or hAMYR3.Additionally or alternatively, compounds of the invention may exhibitimproved solubility in aqueous media, especially at pH values in therange from 4 to 7.5, or at a range of pH values across that range.Moreover, compounds of the present invention may additionally oralternatively exhibit reduced tendency to undergo fibrillation inpharmaceutically relevant aqueous media, especially at pH values in therange from 4 to 7, or at a range of pH values across that range.Furthermore, compounds of the present invention may additionally oralternatively exhibit improved chemical stability (i.e. reduced tendencyto undergo chemical degradation) in aqueous media, especially at pHvalues in the range from 4 to 9, or at a range of pH values across thatrange.

Compounds of the invention may thus be well suited for formulation inacidic media (e.g. pH 4) and in neutral or near-neutral media (e.g. pH 7or 7.4). In contrast to pramlintide, for example, which generallyexhibits poor chemical stability and rapid fibrillation inpharmaceutically relevant aqueous media at neutral pH, compounds of theinvention may be thus well suited for co-formulation with, for example,insulin, various insulin analogues and/or other therapeutic (e.g.anti-diabetic or anti-obesity) agents that require a neutral ornear-neutral formulation pH.

In general it is preferred to use a biological assay which measuresintracellular signalling caused by binding of the compound to therelevant receptor, as discussed above. Activation of thecalcitonin/amylin receptor by compounds of the invention (which behaveas agonists of the receptor) induces cAMP formation and activation ofother intracellular signaling pathways and events. Thus, production ofcAMP or any other suitable parameter in suitable cells expressing thereceptor can be used to monitor agonist activity towards the receptor.

The skilled person will be aware of suitable assay formats, and examplesare provided below. For example, the assays may make use of the humancalcitonin receptor (hCT-R, e.g. isoform 2 of the hCT-R) or the hAMYR3receptor (see the examples below). Where sequences of precursor proteinsare referred to, it should be understood that assays may make use of themature protein, lacking the signal sequence.

EC₅₀ values may be used as a numerical measure of agonist potency at agiven receptor. An EC₅₀ value is a measure of the concentration of acompound required to achieve half of that compound's maximal activity ina particular assay. Thus, for example, a compound having EC₅₀ [hCT-R]lower than the EC₅₀ [hCT-R] of native human amylin, or lower than thatof pramlintide, in a particular assay may be considered to have higherpotency or activity at the receptor than native human amylin, or higherthan that of pramlintide, respectively.

In some embodiments of compounds of the present invention, the EC₅₀towards hCT-R is below 1.5 nM (e.g. 0.001 to 1.5 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hCT-R is below 0.9 nM (e.g. 0.001 to 0.9 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hCT-R is below 0.5 nM (e.g. 0.001 to 0.5 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hCT-R is below 0.3 nM (e.g. 0.001 to 0.3 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hCT-R is below 0.2 nM (e.g. 0.001 to 0.2 nM).

The EC₅₀ at hCT-R may be an indication of the effect of a compound onfood intake, weight gain and/or weight loss. Compounds with lower EC₅₀values at hCT-R may have a greater effect on these parameters.

In some embodiments of compounds of the present invention, the EC₅₀towards hAMYR3 is below 1.0 nM (e.g. 0.001 to 1.0 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hAMYR3 is below 0.5 nM (e.g. 0.001 to 0.5 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hAMYR3 is below 0.4 nM (e.g. 0.001 to 0.4 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hAMYR3 is below 0.3 nM (e.g. 0.001 to 0.3 nM).

In some embodiments of compounds of the present invention, the EC₅₀towards hAMYR3 is below 0.2 nM (e.g. 0.001 to 0.2 nM).

The EC₅₀ at hCT-R (when expressed in the absence of hRAMP1, hRAMP2 andhRAMP3) may be less than the EC₅₀ at any or all of hAMYR1, hAMYR2 andhAMYR3, e.g. at hAMYR3.

For example, the EC₅₀ at hCT-R (when expressed in the absence of hRAMP1,hRAMP2 and hRAMP3) may be less than 10-fold lower than the EC₅₀ at anyor all of hAMYR1, hAMYR2 and hAMYR3, e.g. at hAMYR3.

The EC₅₀ at hCT-R (when expressed in the absence of hRAMP1, hRAMP2 andhRAMP3) may be less than 5-fold lower than the EC₅₀ at any or all ofhAMYR1, hAMYR2 and hAMYR3, e.g. at hAMYR3.

The EC₅₀ at hCT-R (when expressed in the absence of hRAMP1, hRAMP2 andhRAMP3) may be substantially equal to (e.g. +/−50%) the EC₅₀ at any orall of hAMYR1, hAMYR2 and hAMYR3, e.g. at hAMYR3.

The EC₅₀ at hCT-R (when expressed in the absence of hRAMP1, hRAMP2 andhRAMP3) may be higher than the EC₅₀ at any or all of hAMYR1, hAMYR2 andhAMYR3, e.g. at hAMYR3.

Such assays may be performed under the conditions described in Example 2below.

Additionally or alternatively, compounds of the invention may showexcellent resistance to fibrillation. For example, they may show nodetectable fibrillation after 96 hours at pH 4.0 and/or pH 7.0, e.g. at40° C., e.g. under the conditions described in Example 4.

Additionally or alternatively, compounds of the invention may showexcellent chemical stability, i.e. resistance to degradation insolution. For example, they may retain at least 70% purity, at least 75%purity, at least 80% purity, at least 85% purity, at least 90% purity,or at least 95% purity after incubation at pH 4, pH 6, and/or pH 7 at40° C. for 72 hours, or for 14 days, e.g. under the conditions describedin Example 5.

Therapeutic Uses

The compounds of the invention are useful, inter alia, in the reductionof food intake, promotion of weight loss, and inhibition or reduction ofweight gain. They may therefore provide an attractive treatment optionfor, inter alia, obesity and metabolic diseases caused, characterisedby, or associated with, excess body weight.

Thus, the compounds may be used in a method of treating, inhibiting orreducing weight gain, promoting weight loss, reducing food intake,and/or reducing excess body weight. Treatment may be achieved, forexample, by control of appetite, feeding, food intake, calorie intakeand/or energy expenditure.

The compounds may be used in a method of treating obesity as well asassociated diseases, disorders and health conditions, including, but notlimited to, morbid obesity, obesity prior to surgery, obesity-linkedinflammation, obesity-linked gallbladder disease and obesity-inducedsleep apnea and respiratory problems, degeneration of cartilage,osteoarthritis, and reproductive health complications of obesity oroverweight such as infertility.

The compounds may also be used in in a method of prevention or treatmentof Alzheimer's disease, diabetes, type 1 diabetes, type 2 diabetes,pre-diabetes, insulin resistance syndrome, impaired glucose tolerance(IGT), disease states associated with elevated blood glucose levels,metabolic disease including metabolic syndrome, hyperglycemia,hypertension, atherogenic dyslipidemia, hepatic steatosis (“fattyliver”; including non-alcoholic fatty liver disease (NAFLD), whichitself includes non-alcoholic steatohepatitis (NASH)), kidney failure,arteriosclerosis (e.g. atherosclerosis), macrovascular disease,microvascular disease, diabetic heart disease (including diabeticcardiomyopathy and heart failure as a diabetic complication) coronaryheart disease, peripheral artery disease or stroke.

The compounds may also be useful in lowering circulating LDL levelsand/or increasing HDL/LDL ratio.

The effects of the compounds described above may be mediated in whole orin part via an effect on body weight, or may be independent thereof.

Metabolic syndrome is characterized by a group of metabolic risk factorsin one person. They include abdominal obesity (excessive fat tissuearound the abdominal internal organs), atherogenic dyslipidemia (bloodfat disorders including high triglycerides, low HDL cholesterol and/orhigh LDL cholesterol, which foster plaque buildup in artery walls),elevated blood pressure (hypertension), insulin resistance and glucoseintolerance, prothrombotic state (e.g. high fibrinogen or plasminogenactivator inhibitor-1 in the blood), and proinflammatory state (e.g.,elevated C-reactive protein in the blood).

Individuals with metabolic syndrome are at increased risk of coronaryheart disease and other diseases related to other manifestations ofarteriosclerosis (e.g. stroke and peripheral vascular disease). Thedominant underlying risk factor for this syndrome appears to beabdominal obesity.

Pharmaceutical Compositions

The invention also extends to compositions, such as pharmaceuticalcompositions, comprising amylin analogues. As with all aspects of theinvention, it is to be understood that reference to an amylin analogueencompasses reference to pharmaceutically acceptable salts and solvates.

The amylin analogues of the present invention may be formulated aspharmaceutical compositions which are suited for administration with orwithout storage, and which typically comprise a therapeuticallyeffective amount of at least one peptide of the invention, together witha pharmaceutically acceptable carrier, excipient or vehicle.

The term “pharmaceutically acceptable carrier” includes any of thestandard pharmaceutical carriers. Pharmaceutically acceptable carriersfor therapeutic use are well known in the pharmaceutical art and aredescribed, for example, in “Remington's Pharmaceutical Sciences”, 17thedition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa.,USA, 1985. For example, sterile saline and phosphate-buffered saline atslightly acidic or physiological pH may be used. Suitable pH-bufferingagents may, e.g., be phosphate, citrate, acetate,tris(hydroxymethyl)aminomethane (TRIS),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammoniumbicarbonate, diethanolamine, histidine, arginine, lysine or acetate(e.g. as sodium acetate), or mixtures thereof. The term furtherencompasses any carrier agents listed in the US Pharmacopeia for use inanimals, including humans.

A pharmaceutical composition of the invention may be in unit dosageform. In such form, the composition is divided into unit dosescontaining appropriate quantities of the active component or components.The unit dosage form may be presented as a packaged preparation, thepackage containing discrete quantities of the preparation, for example,packaged tablets, capsules or powders in vials or ampoules. The unitdosage form may also be, e.g., a capsule, cachet or tablet in itself, orit may be an appropriate number of any of these packaged forms. A unitdosage form may also be provided in single-dose injectable form, forexample in the form of a pen device containing a liquid-phase (typicallyaqueous) composition. Compositions may be formulated for any suitableroute and means of administration. Pharmaceutically acceptable carriersor diluents include those used in formulations suitable for e.g. oral,intravitreal, rectal, vaginal, nasal, topical, enteral or parenteral(including subcutaneous (sc), intramuscular (im), intravenous (iv),intradermal and transdermal) administration or administration byinhalation. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmaceutical formulation.

Subcutaneous or transdermal modes of administration may in some cases besuitable for peptides of the invention.

Further embodiments relate to devices, dosage forms and packages used todeliver the pharmaceutical formulations of the present invention. Thus,at least one peptide in a stable or preserved formulation or solutiondescribed herein can be administered to a patient in accordance with thepresent invention via a variety of delivery methods, including by sc orim injection, or by transdermal, pulmonary or transmucosaladministration, or by implant, or by use of an osmotic pump, cartridge,micro-pump or other means recognized by a person of skill in the art.

Still further embodiments relate to oral formulations and oraladministration. Formulations for oral administration may rely on theco-administration of adjuvants (e.g. resorcinols and/or nonionicsurfactants such as polyoxyethylene oleyl ether andn-hexadecylpolyethylene ether) to artificially increase the permeabilityof the intestinal walls, and/or the co-administration of enzymaticinhibitors (e.g. pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) or trasylol) to inhibit enzymaticdegradation. The active constituent compound of a solid-type dosage formfor oral administration can be mixed with at least one additive, such assucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol,dextran, starches, agar, alginates, chitins, chitosans, pectins, gumtragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic orsemisynthetic polymer, or glyceride. These dosage forms can also containother type(s) of additives, e.g. an inactive diluting agent, a lubricant(such as magnesium stearate), a paraben, a preserving agent (such assorbic acid, ascorbic acid or alpha-tocopherol), an antioxidant (such ascysteine),a disintegrant, binder, thickener, buffering agent,pH-adjusting agent, sweetening agent, flavoring agent or perfumingagent.

Dosages

A typical dosage of an amylin analogue as employed in the context of thepresent invention may be in the range from about 0.0001 to about 100mg/kg body weight per day, such as from about 0.0005 to about 50 mg/kgbody weight per day, such as from about 0.001 to about 10 mg/kg bodyweight per day, e.g. from about 0.01 to about 1 mg/kg body weight perday, administered in one or more doses, such as from one to three doses.The exact dosage employed will depend, inter alia, on: the nature andseverity of the disease or disorder to be treated, on the sex, age, bodyweight and general condition of the subject to be treated, on possibleother, concomitant, disease or disorder that is undergoing or is toundergo treatment, as well as on other factors that will be known to amedical practitioner of skill in the art.

An amylin analogue of the invention may be administered continuously(e.g. by intravenous administration or another continuous drugadministration method), or may be administered to a subject atintervals, typically at regular time intervals, depending on the desireddosage and the pharmaceutical composition selected by the skilledpractitioner for the particular subject. Regular administration dosingintervals include, e.g., once daily, twice daily, once every two, three,four, five or six days, once or twice weekly, once or twice monthly, andthe like. Such regular peptide administration regimens may, in certaincircumstances such as, e.g., during chronic long-term administration, beadvantageously interrupted for a period of time so that the medicatedsubject reduces the level of, or stops taking, the medication, oftenreferred to as taking a “drug holiday.” Drug holidays are useful for,e.g., maintaining or regaining sensitivity to a drug especially duringlong-term chronic treatment, or to reduce unwanted side-effects oflong-term chronic treatment of the subject with the drug. The timing ofa drug holiday depends on the timing of the regular dosing regimen andthe purpose for taking the drug holiday (e.g., to regain drugsensitivity and/or to reduce unwanted side effects of continuous,long-term administration). In some embodiments, the drug holiday may bea reduction in the dosage of the drug (e.g. to below the therapeuticallyeffective amount for a certain interval of time). In other embodiments,administration of the drug is stopped for a certain interval of timebefore administration is started again using the same or a differentdosing regimen (e.g. at a lower or higher dose and/or frequency ofadministration). A drug holiday of the invention may thus be selectedfrom a wide range of time-periods and dosage regimens. An exemplary drugholiday is two or more days, one or more weeks, or one or more months,up to about 24 months of drug holiday. So, for example, a regular dailydosing regimen with a peptide of the invention may, for example, beinterrupted by a drug holiday of a week, or two weeks, or four weeks,after which time the preceding, regular dosage regimen (e.g. a daily ora weekly dosing regimen) is resumed. A variety of other drug holidayregimens are envisioned to be useful for administering peptides of theinvention.

Thus, the peptide may be delivered via an administration regime whichcomprises two or more administration phases separated by respective drugholiday phases.

During each administration phase, the peptide is administered to therecipient subject in a therapeutically effective amount according to apre-determined administration pattern. The administration pattern maycomprise continuous administration of the drug to the recipient subjectover the duration of the administration phase. Alternatively, theadministration pattern may comprise administration of a plurality ofdoses of the peptide to the recipient subject, wherein said doses arespaced by dosing intervals.

A dosing pattern may comprise at least two doses per administrationphase, at least five doses per administration phase, at least 10 dosesper administration phase, at least 20 doses per administration phase, atleast 30 doses per administration phase, or more.

Said dosing intervals may be regular dosing intervals, which may be asset out above, including once daily, twice daily, once every two, three,four, five or six days, once or twice weekly, once or twice monthly, ora regular and even less frequent dosing interval, depending on theparticular dosage formulation, bioavailability, and pharmacokineticprofile of the peptide.

An administration phase may have a duration of at least two days, atleast a week, at least 2 weeks, at least 4 weeks, at least a month, atleast 2 months, at least 3 months, at least 6 months, or more.

Where an administration pattern comprises a plurality of doses, theduration of a possible following drug holiday phase is longer than thedosing interval used in that administration pattern. Where the dosinginterval is irregular, the duration of a drug holiday phase may begreater than the mean interval between doses over the course of theadministration phase. Alternatively the duration of the drug holiday maybe longer than the longest interval between consecutive doses during theadministration phase.

The duration of a possible drug holiday phase may be at least twice thatof the relevant dosing interval (or mean thereof), at least 3 times, atleast 4 times, at least 5 times, at least 10 times, or at least 20 timesthat of the relevant dosing interval or mean thereof.

Within these constraints, a drug holiday phase may have a duration of atleast two days, at least a week, at least 2 weeks, at least 4 weeks, atleast a month, at least 2 months, at least 3 months, at least 6 months,or more, depending on the administration pattern during the previousadministration phase.

An administration regime entailing the use of drug holiday comprises atleast 2 administration phases. Consecutive administration phases areseparated by respective drug holiday phases. Thus the administrationregime may comprise at least 3, at least 4, at least 5, at least 10, atleast 15, at least 20, at least 25, or at least 30 administrationphases, or more, each separated by respective drug holiday phases.

Consecutive administration phases may utilise the same administrationpattern, although this may not always be desirable or necessary.However, if other drugs or active agents are administered in combinationwith a peptide of the invention, then typically the same combination ofdrugs or active agents is given in consecutive administration phases. Incertain embodiments, the recipient subject is a human.

Combination Therapy

An amylin analogue of the invention may be administered as part of acombination therapy together with another active agent for the treatmentof the disease or disorder in question, e.g. an anti-diabetic agent, ananti-obesity agent, an agent for treatment of metabolic syndrome, ananti-dyslipidemia agent, an anti-hypertensive agent, a proton pumpinhibitor, or an anti-inflammatory agent. In such cases, the two activeagents may be given together or separately, e.g. as constituents in thesame pharmaceutical composition or formulation, or as separateformulations.

Thus a peptide of the invention may have some benefit if administered incombination with an anti-diabetic agent of known type, including, butnot limited to, metformin, a sulfonylurea, a glinide, a DPP-IVinhibitor, a glitazone, a GLP-1 receptor agonist (including GLP-1 or aGLP-1 analogue, an exendin-4 or an exendin-4 analogue, any other GLP-1receptor agonist including liraglutide (Saxenda™, Victoza™), Dulaglutideor Albiglutide or a glucagon-GLP-1 dual agonist, e.g. as described inWO2008/101017, WO2008/152403, WO2010/070252, WO2010/070253,WO2010/070255, WO2010/070251, WO2011/006497, WO2011/160630,WO2011/160633, WO2013/092703, WO2014/041195, WO2015/055801,WO2015/055802), an SGLT2 inhibitor (i.e. an inhibitor of sodium-glucosetransport, e.g. a gliflozin such as empagliflozin, canagliflozin,dapagliflozin or ipragliflozin), a GPR40 agonist (FFAR1/FFA1 agonist,e.g. fasiglifam), or an insulin or an insulin analogue. Examples ofappropriate insulin analogues include, but are not limited to, Lantus™,Novorapid™, Humalog™, Novomix™, Actraphane™ HM, Levemir™ Degludec™ andApidra™. Other relevant anti-diabetic agents in this connection includeGLP-1 receptor agonists, such as exenatide (Byetta™ and Bydureon™exendin-4) and Byetta LAR™, lixisenatide (Lyxumia™) and liraglutide(Victoza™).

Moreover, a peptide of the invention may be used in combination with ananti-obesity agent of known type, including, but not limited to, peptideYY or an analogue thereof, neuropeptide Y (NPY) or an analogue thereof,a cannabinoid receptor 1 antagonist, a lipase inhibitor, Human prolsletPeptide (HIP), a melanocortin receptor 4 agonist, a GLP-1 receptoragonist (including GLP-1 or a GLP-1 analogue, an exendin-4 or anexendin-4 analogue, any other GLP-1 receptor agonist includingliraglutide (Saxenda™, Victoza™), Dulaglutide or Albiglutide or aglucagon-GLP-1 dual agonist, e.g. as described in WO2008/101017,WO2008/152403, WO2010/070252, WO2010/070253, WO2010/070255,WO2010/070251, WO2011/006497, WO2011/160630, WO2011/160633,WO2013/092703, WO2014/041195, WO2015/055801, WO2015/055802), Orlistat™,Sibutramine™, phentermine, a melanin concentrating hormone receptor 1antagonist, CCK, amylin, pramlintide and leptin, as well as analoguesthereof.

A peptide of the invention may further be used in combination with ananti-hypertension agent of a known type, including, but not limited to,an angiotensin-converting enzyme inhibitor, an angiotensin II receptorblocker, a diuretic, a beta-blocker or a calcium channel blocker.

A peptide of the invention may still further be used in combination withan anti-dyslipidemia agent of known type, including, but not limited to,a statin, a fibrate, a niacin, a PSCK9 (Proprotein convertasesubtilisin/kexin type 9) inhibitor, or a cholesterol absorptioninhibitor.

A peptide of the invention may also be used in combination with a protonpump inhibitor (i.e. a pharmaceutical agent possessing pharmacologicalactivity as an inhibitor of H⁺/K⁺-ATPase) of known type, including, butnot limited to, an agent of the benzimidazole derivative type or of theimidazopyridine derivative type, such as Omeprazole™, Lansoprazole™,Dexlansoprazole™, Esomeprazole™, Pantoprazole™, Rabeprazole™, Zolpidem™,Alpidem™, Saripidem™ or Necopidem™.

In addition, with regard to anti-inflammatory treatment, a peptide ofthe invention may be beneficial if administered in combination with ananti-inflammatory agent of known type, including, but not limited to:

steroids and corticosteroids, such as beclomethasone,methylprednisolone, betamethasone, prednisone, dexamethasone, andhydrocortisone;

non-steroidal anti-inflammatory agents (NSAIDs), such as propionic acidderivatives (e.g. alminoprofen, benoxaprofen, bucloxic acid, carprofen,fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen,ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen,suprofen, tiaprofenic acid and tioxaprofen); acetic acid derivatives(e.g. indomethacin, acemetacin, alclofenac, clidanac, diclofenac,fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac,oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac);fenamic acid derivatives (e.g. flufenamic acid, meclofenamic acid,mefenamic acid, niflumic acid and tolfenamic acid); biphenylcarboxylicacid derivatives (e.g. diflunisal and flufenisal); oxicams (e.g.isoxicam, piroxicam, sudoxicam and tenoxicam); salicylates (e.g.acetylsalicylic acid and sulfasalazine); and pyrazolones (e.g. apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone andphenylbutazone);

COX II inhibitors, such as rofecoxib and celecoxib; preparations ofinterferon beta (e.g. interferon beta-1a or interferon beta-1b);

and certain other compounds, such as 5-aminosalicylic acid and prodrugsand pharmaceutically acceptable salts thereof.

Metformin has also been demonstrated to have anti-inflammatoryproperties (see, e.g., Haffner et al., Diabetes 54: 1566-1572 (2005))and as such may also be useful in combination with compounds (peptides)of the invention.

Devices and Kits

In some embodiments, the invention relates to a device comprising anamylin analogue or pharmaceutical composition of the invention, fordelivery of the analogue to a subject. Via such devices, amylinanalogues can be administered to a patient via a variety of deliverymethods, including: intravenous, subcutaneous, intramuscular orintraperitoneal injection; oral administration; transdermaladministration; pulmonary or transmucosal administration; administrationby implant, osmotic pump, cartridge or micro pump; or by other meansrecognized by a person of skill in the art.

In some embodiments, the invention relates to a kit comprising an amylinanalogue of the invention or a pharmaceutical composition of theinvention. In certain embodiments, the kit further comprises packagingand/or instructions for use.

The device or kit may be useful for combination therapy as describedabove. Thus the device or kit may further comprise a further activeagent, e.g. an anti-diabetic agent, an anti-obesity agent, an agent fortreatment of metabolic syndrome, an anti-dyslipidemia agent, ananti-hypertensive agent, a proton pump inhibitor, or ananti-inflammatory agent as described above, or a pharmaceuticalcomposition comprising such an active agent.

EXAMPLES

The following examples demonstrate certain specific embodiments of thepresent invention. The following examples were carried out usingstandard techniques that are well known and routine to those of skill inthe art, except where otherwise described in detail. It is to beunderstood that these examples are for illustrative purposes only and donot purport to be wholly definitive as to conditions or scope of theinvention. As such, they should not be construed as limiting the scopeof the present invention in any way.

Abbreviations employed in the examples include:

-   -   Acm: acetaminomethyl    -   Alloc: allyloxycarbonyl    -   Boc: tert-butoxycarbonyl    -   BSA: bovine serum albumin    -   cAMP: cyclic adenosine monophosphate    -   COMU™:        (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium        hexafluorophosphate    -   DCM: dichloromethane    -   DIPEA: diisopropylethylamine    -   DMEM: Dulbecco's Modified Eagle Medium    -   DMF: N,N-dimethylformamide    -   DODT: 3,6-dioxa-1,8-octanedithiol    -   ESI-MS: electron spray ionization mass spectrometry    -   EtOH: ethanol    -   Et₂O: diethyl ether    -   FCS: fetal calf serum    -   Fmoc: 9-fluorenylmethoxycarbonyl    -   HATU: 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        hexafluorophosphate    -   HEPES: N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid    -   HPLC: high performance liquid chromatography    -   IBMX: 3-isobutyl-1-methylxanthine    -   MeCN: acetonitrile    -   MS: mass spectrometry    -   NEP: N-ethylpyrrolidone    -   NMP N-methylpyrrolidone    -   OAll: allylester    -   PBS: phosphate-buffered saline    -   p-ERK: phosphorylated extracellular regulated kinase    -   RP-HPLC: reverse phase high performance liquid chromatography    -   TFA: trifluoroacetic acid    -   TIS: triisopropylsilane    -   Trt: trityl (i.e. triphenylmethyl)    -   v/v: volume/volume    -   w/v: weight/volume

The following examples are provided to illustrate certain embodiments ofthe invention and are not intended to limit the scope of the invention.

Measurement of Physiological Parameters

Unless otherwise specified, whole-blood glucose levels were determinedon tail-vein blood samples by the Biosen (EKF Diagnostic, Germany)enzyme-based electrode method. Blood samples were analyzed for glycatedhemoglobin (HbA1c) using a Cobas c111 analyzer (Roche Diagnostics,Mannheim, Germany).

Example 1 Synthesis of Compounds

The following compounds were synthesised:

(Compound 1) (SEQ ID NO: 55)[19CD]-isoGlu-RD()GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 2) (SEQ ID NO: 56)[19CD]-isoGlu-RD()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 3) (SEQ ID NO: 57)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 4) (SEQ ID NO: 58)[19CD]-isoGlu-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 5) (SEQ ID NO: 59)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 6) (SEQ ID NO: 60)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 7) (SEQ ID NO: 61)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLHRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNTP-NH₂ (Compound 8) (SEQ ID NO: 62)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 9) (SEQ ID NO: 63)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 10) (SEQ ID NO: 64)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLARFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 11) (SEQ ID NO: 65)[19CD]-isoGlu-ED()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 12) (SEQ ID NO: 66)[19CD]-isoGlu-RD()GEATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 13) (SEQ ID NO: 67)[19CD]-isoGlu-RD()GTLTK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 14) (SEQ ID NO: 68)[19CD]-isoGlu-RD()GTASK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 15) (SEQ ID NO: 69)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 16) (SEQ ID NO: 70)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 17) (SEQ ID NO: 71)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 18) (SEQ ID NO: 72)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 19) (SEQ ID NO: 73)[19CD]-isoGlu-RD()GTAT-hLys()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 20) (SEQ ID NO: 74)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 21) (SEQ ID NO: 75)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 22) (SEQ ID NO: 76)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLTRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSST-Hyp-NH₂ (Compound 23) (SEQ ID NO: 77)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 24) (SEQ ID NO: 78)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 25) (SEQ ID NO: 79)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRATF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 26) (SEQ ID NO: 80)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 27) (SEQ ID NO: 81)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 28) (SEQ ID NO: 82)[19CD]-isoGlu-RD()QTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 29) (SEQ ID NO: 83)[19CD]-isoGlu-RD()PTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 30) (SEQ ID NO: 84)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 31) (SEQ ID NO: 85)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 32) (SEQ ID NO: 86)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRGGF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 33) (SEQ ID NO: 87)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRANF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 34) (SEQ ID NO: 88)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 35) (SEQ ID NO: 89)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 36) (SEQ ID NO: 90)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 37) (SEQ ID NO: 91)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSFGly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 38) (SEQ ID NO: 92)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLORTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 39) (SEQ ID NO: 93)[19CD]-isoGlu-KD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-AIle(Me)-LSSTEVGSNTHyp-NH₂ (Compound 40) (SEQ ID NO: 94)[19CD]-isoGlu-RD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 41) (SEQ ID NO: 95)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 42) (SEQ ID NO: 96)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 43) (SEQ ID NO: 97)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 44) (SEQ ID NO: 98)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 45) (SEQ ID NO: 99)[19CD]-isoGlu-R-Dap()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 46) (SEQ ID NO: 100)[19CD]-isoGlu-R-Dab()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 47) (SEQ ID NO: 101)[19CD]-isoGlu-R-Orn()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 48) (SEQ ID NO: 102)[19CD]-isoGlu-R-Dap()-GTAT-Aad()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 49) (SEQ ID NO: 103)[19CD]-isoGlu-R-Dab()-GTATE()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 50) (SEQ ID NO: 104)[19CD]-isoGlu-R-Aad()-GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 51) (SEQ ID NO: 105)[19CD]-isoGlu-RE()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂

For comparison purposes, three compounds having disulphide bridges(instead of lactam bridges) and two uncyclised compounds weresynthesised:

Ref. 1 (SEQ ID NO: 106)[19CD]-isoGlu-RDGTAT-Orn-ATERLAHFLQRSSF-Sar-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ Ref. 2 (SEQ ID NO: 107)[19CD]-isoGlu-RC()GTATC()ATERLAHFLQRSSF-Sar-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ Ref. 3 (SEQ ID NO: 108)[19CD]-isoGlu-RDGTAT-Orn-ATERLA-Aad-FLQRSSF-Sar-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ Ref. 4 (SEQ ID NO: 109)[19CD]-isoGlu-RC()GTATC()ATERLA-Aad-FLQRSSF-Sar-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ Ref. 5 (SEQ ID NO: 110)[19CD]-isoGlu-RC()NTATC()ATQRLADFLQRSSF-Sar-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂

A further reference compound, designated NN96, is{N-α-[(S)-4-carboxy-4-(19-carboxynonadecanoylamino)butyryl]-[Arg1,Glu14,His17,Pro37]-pramlintide;disclosed as Example 96 in WO 2012/168430 A2, and having the amino acidsequence

RC()NTATC()ATQRLAEFLHHSSNNFGPILPPTNVGSNTP.

Parentheses “( )” indicate intramolecular lactam bridges (or disulphidebridges, where appropriate) formed between the side chains of theresidues at positions 2 and 7 of the relevant amino acid sequences.

Unless otherwise specified, reagents and solvents employed in thefollowing were available commercially in standard laboratory reagent oranalytical grade, and were used without further purification.

General Procedures for Solid-Phase Synthesis of Peptides

A CEM Liberty Peptide Synthesizer or a CEM Liberty Blue PeptideSynthesizer was employed, using standard Fmoc chemistry. TentaGel™ S Ramresin (1 g; 0.25 mmol/g) was swelled in DMF (10 ml) prior to use andtransferred between tube and reaction vessel using DCM and DMF.Pseudoprolines [i.e. dipeptides employed to minimize aggregation duringpeptide synthesis, such as Fmoc-Phe-Thr(ψ-Me,Me-Pro)-OH andFmoc-Asp-Ser(ψ-Me,Me-Pro)-OH and Fmoc-Ser-Ser(ψ-Me,Me-Pro)-OH] were usedwhere appropriate, and non-naturally occurring amino acids and othersuitable building blocks were employed without any changes to thegeneral procedure.

The following optical isomers of particular amino acids (includingnon-naturally occurring amino acids) were employed in the synthesis ofthe compounds:

-   -   Hyp: (2S,4R)-4-hydroxyproline [also denoted        (4R)-4-hydroxy-L-proline].    -   Aad: (2S)-2-aminoadipic acid    -   Dab: (2S)-2,4-diaminobutanoic acid    -   Dap: (2S)-2,3-diaminopropanoic acid    -   hLys: (2S)-2-amino-7-amino-heptanoic acid, also known as        homo-lysine    -   Gly(Me): N-methylglycine, also known as sarcosine    -   Ile(Me): N-methylisoleucine

Coupling:

CEM Liberty Peptide Synthesizer: an Fmoc-amino acid in DMF/DCM (2:1; 0.2M; 5 mL) was added to the resin in a CEM Discover microwave unittogether with COMU/DMF (0.5 M; 2 mL) and DIPEA/NMP (2.0 M; 1 mL). Thecoupling mixture was heated to 75° C. for 5 min while nitrogen wasbubbled through the mixture. The resin was then washed with DMF (4×10mL). Alternatively the coupling was done without heating and thereaction time extended to 60 min in this case.

CEM Liberty Blue Peptide Synthesizer: an Fmoc-amino acid in DMF (0.2 M;5 mL) was added to the resin in a CEM Discover microwave unit togetherwith DIC/DMF (0.5 M; 2 mL) and Oxyma/DMF (2.0 M; 1 mL). The couplingmixture was heated to 90° C. for 2 min while nitrogen was bubbledthrough the mixture. The resin was then washed with DMF (4×10 mL).Alternatively the coupling was done without heating and the reactiontime extended to 60 min in this case.

Independent of CEM Synthesizer type, in the case of difficult couplings(e.g. coupling of a residue immediately after an N-methylated amino acidresidue or other sterically hindered amino acid residue as recognized bya person of skill in the art) the coupling was repeated one or moretimes.

Deprotection:

Piperidine/DMF (1:4, i.e. 1 part piperidine to 4 parts DMF by volume; 10mL) was added to the resin for initial deprotection, and the mixture wasmicrowave-heated (40° C.; 30 sec). The reaction vessel was drained and asecond portion of piperidine/DMF (1:4; 10 mL) was added and heated (75°C.; 3 min) again. The resin was then washed with DMF (6×10 mL).

Oxidative Cyclisation

Intramolecular ring formation (disulfide bridge formation) between Cysresidues in positions 2 and 7 (initially coupled in the form ofAcm-protected cysteines) was performed with the peptide still attachedto the resin, using 163 mg thallium(III) trifluoroacetate [TI(TFA)₃] in5 mL NMP in a simultaneous Acm-deprotection and disulfide-formationstep. (alternative method: addition of 10 eq of iodine to a 50 mMsolution of resin bound peptide in acetic acid and stirring for 18-24 h)

Lactam Cyclisation

The following procedure for the coupling of Asp and Lys isrepresentative for all lactam formations where the amino acid side chaincontaining the carboxyl-function is protected with OAll and the aminoacid side chain containing the amino group is Alloc-protected. Afterassembly of the full peptide sequence, deprotection of Asp(OAll) inposition 2 and Lys(Alloc) in position 7 was performed using 29 mgtetrakis(triphenylphosphine)palladium(0) and phenylsilane 125 μL in 20mL DCM. Subsequently, the lactam bridge was formed between the Aspresidue (2) and Lys residue (7) using 414 mg HCTU and 346 μL DIPEA in 20mL DMF. Both steps were performed with the peptide still attached to theresin.

Cleavage:

The resin was washed with EtOH (3×10 mL) and Et₂O (3×10 mL) and dried toconstant weight at room temperature (r.t.). The crude peptide wascleaved from the resin by treatment with TFA/TIS/H₂O (90:5:5; 40 mL; 2h; r.t.) or alternatively with TFA/DODT (95:5; 40 mL; 2 h; r.t.). Mostof the TFA was removed under reduced pressure, and the crude peptide wasprecipitated and washed three times with Et₂O and dried to constantweight at room temperature.

Purification and Characterisation:

The crude peptide was purified by preparative reverse phase HPLC using agradient of solvent A (0.1% aqueous TFA) and solvent B (0.1% TFA, 90%MeCN in water) on either a PerSeptive Biosystems VISION Workstation or aGilson system (Pumps: “Pump 305”, “331 Pump”, “332 Pump”, “402 SyringePump”; column changer “Valvemate® II”UV detector “UV/Vis-155”; and thefraction collector “GX 281” equipped with a suitable column and afraction collector) or a Waters Autopurification HPLC/MS System (2525pump System, Waters 2996 DAD, sample manager Waters 2767, MS ZQ singlequadrupole. Columns: XSelect CSH 130 Prep C18 5 mm ODB 30×150 mm orKinetex 5 mm C8 100A 150×21.2 mm). Fractions were analysed by analyticalHPLC and MS, and relevant fractions were pooled and lyophilised. Thefinal product was characterized by HPLC and MS.

One of skill in the art will appreciate that standard methods of peptidesynthesis may be used to generate the compounds of the invention.

Example 2

hCT-R and hAMYR3 Assays

For the assessment of the in vitro activity of test peptides, cell linesexpressing recombinant human calcitonin receptor (hCT-R) or recombinanthuman amylin 3 receptor (hAMYR3) in the background of the humanastrocytoma cell line 1321N1 were purchased from DiscoveRx Corporation(Cat. No. 95-0161C6 and 95-0166C6). The hAMYR3 is a hetero-oligomer ofthe calcitonin receptor (Isoform 2; Gene ID 799) and RAMPS (Gene ID10268) that forms when both genes are expressed in the same cell. ThehCT-R cell line expresses only the recombinant human calcitonin receptorgene (Isoform 2; Gene ID 799). Upon activation of hCT-R or hAMYR3 by thetest peptides the formation of cAMP is induced and was measured usingthe AlphaScreen® cAMP Assay kit from Perkin-Elmer (Cat. No. 6760635R).

Briefly, 1321N1 cells expressing hCT-R or hAMYR3 were seeded in 384-wellmicrotiter plates (Falcon Optilux White, Cat.-No. 10448642) at 5,000cells in 50 μL growth medium per well (AssayComplete 1321N1 Cell CultureKit, DiscoveRx Corp.), and incubated for 24 hat 37° C., 5% CO2. On theday of analysis growth medium was removed and the cells were stimulatedby adding 10 μL of stimulation buffer (10 nM Hepes pH 7.4, 140 nM NaCl,3.6 nM KCl, 0.5 nM NaH₂PO₄, 0.5nM MgSO₄, 1.5 nM CaCl₂, 5 nM NaHCO₃, 0.5nM IBMX, 0.1% BSA) containing increasing concentrations of testpeptides, and incubation for 45 min at room temperature. The stimulationwas stopped by adding 5.6 μL/well donor bead detection mix (5 mM HepespH7.4, 0.5% TWEEN 20, 0.1% BSA, 0.05 mg/mL donor beads, 62.5 nMbiotinylated cAMP) and 4.5 μL/well acceptor bead solution (5 mM HepespH7.4, 0.5% TWEEN 20, 0.1% BSA, 0.05 mg/mL acceptor beads). Afterthorough mixing the plates were incubated in the dark for 1 h at roomtemperature and the cAMP content of the resulting cell lysates wasestimated according to the AlphaScreen® cAMP Assay manufacturer'sinstructions. EC50 values were estimated by computer-aided curve fittingof results of at least 7 different compound concentrations.

The in vitro activity results (expressed as EC₅₀ values) are summarizedin Table 1, below.

Example 3

Solubility Determination

Test peptide is weighed into a suitable vial and the respective buffer(acetate buffer pH 4.0, phosphate buffer pH 6, histidine buffer pH 6 and7; all at 40 nM concentration) added to a total volume of 0.5 mL.

Vials are shaken at room temperature for 2 h and filtered through a 0.45μm filter. The resulting solutions are analyzed by RP-HPLC on a C18column with gradient elution using a formic acid/acetonitrile/watereluent system. The area of the main peak is determined by UVspectroscopy at 230 nm at each sampling time point. The dissolvedconcentration is calculated by an external standard method.

Example 4

Assessment of Physical Stability

Aggregation in the form of fibril formation was detected using theamyloid-specific dye Thioflavin T (ThT), which is frequently employed todemonstrate the presence of fibrils in solution (see, e.g., Groenning,M., J. Chem. Biol. 3(1) (2010), pp. 1-18; Groenning et al., J. Struct.Biol. 158 (2007) pp. 358-369; and Levine, H., III, Protein Sci. 2 (1993)pp. 404-410) Test peptides (2 mg/mL) were dissolved in demineralizedwater adjusted to pH 2.5 with HCl, at ambient temperature (typically 25°C.). Solutions containing (i) 1 mg/mL of test peptide, 40 μM ThT and 50mM phosphate (Ph) buffer (pH 7.0), (ii) 1 mg/mL of test peptide, 40 μMThT and 50 mM histidine (His) buffer (pH 7.0), and (iii) 1 mg/mL of testpeptide, 40 μM ThT and 50 mM acetate (Ac) buffer (pH 4.0), were loadedin a 96-well black fluorescence plate (clear bottom) in triplicate. Datawere collected at fixed intervals of 10 min, each preceded by 300 s ofautomixing (agitation), over a period of 96 hours at 40° C. Physicalstability is determined by measuring the fluorescence intensity overtime. A significant increase in intensity is rated as fibrillationdetected (FD). Data are summarized in Table 1 below.

Example 5

Assessment of Chemical Stability

Samples of each test peptide were dissolved in acetate buffer pH 4 andphosphate buffer pH 6 and 7 (all buffer 40 mM). The final peptideconcentration was 1 mg/mL. Samples were placed in glass vials andincubated at 40° C. The samples were analyzed by RP-HPLC on a C8 columnwith gradient elution using a trifluoroacetic acid/acetonitrile/watereluent system. The area-percentage (area-%) of the main peak wasdetermined by UV spectroscopy at 220 nm at each sampling time point.

The % degradation was calculated by subtracting the main peak areapercentage at start (t=0) from the main peak area percentage at eachsampling time point.

The results of the chemical stability assessment after 3 days ofincubation time are summarized as % degradation in Table 1 (below).

The results of the chemical stability assessment after 14 days ofincubation time are summarized as % degradation in Table 2 (below).

In an alternative analysis, peptides were dissolved in MilliQ water andpH adjusted to 4.0, 7.5 or 9.0. Samples were placed in glass vials andincubated at 40° C. The samples were analyzed by RP-HPLC on a C8 columnwith gradient elution using a trifluoroacetic acid/acetonitrile/watereluent system. The area-percentage (area-%) of the main peak wasdetermined by UV spectroscopy at 220 nm at each sampling time point.

The % degradation was calculated by subtracting the main peak areapercentage at start (t=0) from the main peak area percentage at eachsampling time point.

The results of the chemical stability assessment after 8 days ofincubation time are summarized as % degradation in Table 3 (below).

TABLE 1 EC₅₀, chemical stability and fibrillation data hCT-R hAMYR3Fibrillation* (in buffer) % degradation after 72 h at 40° C. EC50 EC50pH 4 pH 7 pH 7 pH 4 pH 6 pH 7 Compound no. [nM] [nM] acetate histidinephosphate acetate phosphate phosphate 1 13.6 9.3 2 0.105 0.032 3 0.0810.035 FND FND FD <1 3.2 1.2 4 0.160 0.092 FND FND FND <1 <1 <1 5 0.1140.032 6 0.327 0.011 7 0.289 0.032 8 0.129 0.059 FND FND FND <1 <1 <1 90.027 0.013 10 0.345 0.068 FD FND FND <1 <1 <1 11 0.068 0.024 FND FNDFND <1 <1 <1 12 0.893 0.999 13 1.411 0.456 14 0.386 0.108 15 0.056 0.017FND FD FD <1 <1 <1 16 0.043 0.035 17 0.058 0.025 FND FND FND 1.2 <1 <118 0.085 0.031 FND FND FND <1 <1 <1 19 0.028 0.006 20 0.009 0.003 FD FNDFND 1.0 1.3 2.0 21 0.037 0.010 FND FND FND <1 <1 <1 22 0.474 0.051 231.931 0.706 24 0.038 0.013 25 0.844 0.336 FND FND FND — — <1 26 0.7690.120 27 0.521 0.325 FND FND FND <1 <1 1.7 28 1.229 0.268 29 1.070 0.31230 0.232 0.122 FD FND FND 31 0.227 0.066 32 0.006 0.005 FD FND FND 1.3<1 2.2 33 0.019 0.009 FND FND FND <1 1 <1 34 0.206 0.063 FND FND FND <1<1 2.8 35 0.066 0.028 FND FND FND <1 <1 1.4 36 0.026 0.007 FD FND FND2.5 2.1 1.0 37 0.116 0.031 FD FND FND — <1 3.0 38 0.114 0.060 FD FND FND<1 <1 <1 39 0.094 0.024 FD FND FND <1 <1 <1 40 0.028 0.008 FD FND FND1.1 <1 <1 41 0.095 0.024 FND FND FND <1 <1 <1 42 0.006 0.003 43 0.0210.007 FD FD FD 44 0.020 0.009 FD FND FND 45 299 279 46 7.5 5.5 47 10.87.7 48 0.107 0.020 49 1.038 0.442 50 3.3 1.5 51 0.861 0.306 Ref. 1 20.317.1 Ref. 2 0.019 0.003 Ref. 3 18.2 13.2 Ref. 4 0.075 0.031 FND FND FNDRef. 5 0.063 0.028 FD FND FND *FND = fibrillation not detected; FD =fibrillation detected

TABLE 2 Chemical stability % degradation after 14 days at 40° C. pH 4 pH6 pH 7 Compound no. acetate phosphate phosphate 32 <1 <1 <1 41 1.1 <1 <110 1.9 1.8 9.1 14 2.2 <1 2.6 18 <1 1.0 1.9 37 ND <1 <1 36 ND 1.6 <1  41.6 <1 6.4 38 ND ND <1 20 ND <1 3.7 16 ND <1 ND  3 ND 1.0 ND 35 2 <1 <1Ref. 2 <1 4.6 ND Ref. 4 3.2 12.6 20.3 Ref. 5 3.0 5.1 12.7 NN96 3.0 10.133.2 ND—not determined

TABLE 3 Chemical stability % degradation after 8 days at 40° C. Compoundno. pH 4 pH 7.5 pH 9 51 3.1 4.6 4.6 49 1.7 2.1 3.0 48 1.8 2.3 2.5 19 3.96.2 4.2

Example 6

Pharmacokinetic (PK) Profiling in Rats

Sprague Dawley male rats were given a single subcutaneous (sc) bolus ofeach peptide to be tested, as specified below.

30 nmol/kg doses of compound were administered. Blood samples were drawnfrom the tail vein prior to dosing and at 24 and 96 hours after dosing.The rats were euthanized immediately after the last blood sampling byconcussion and cervical dislocation.

The dosing vehicle used for each test peptide was a mannitol-containinghistidine buffer (pH 7.0). Plasma samples were analyzed afterprecipitation with ethanol by liquid chromatography mass spectrometry(LC-MS/MS). Mean plasma concentrations were used for calculation of thepharmacokinetic parameters.

Plasma terminal elimination half-life (t_(1/2)) was determined asln(2)/λz, where λz is the magnitude of the slope of the log linearregression of the log concentration versus time profile during theterminal phase.

Results

The plasma terminal elimination half-lives (t_(1/2)) for all testedpeptides were determined to be in the range of 21.3 h to 36.1 h.

TABLE 4 in vivo half life Compound No. t_(1/2) [h] 3 21.3 4 31.9 18 30.120 31.1 35 36.1 38 31.8

Example 7

Effect on Acute Food Intake and Body Weight in Normal Sprague DawleyRats

Sprague Dawley (SD) rats were obtained from Janvier Labs, France. Theanimals arrived at least 14 days before the study start to allowacclimatization to experimental conditions. From arrival and throughoutthe study, the rats were housed in groups of 2 to 4 (n=2-4) in a light-,temperature- and humidity-controlled room. Animals had access ad libitumto food (KLIBA 3430, Provimi Kliba AG, Switzerland) and water (domesticquality tap water) during the entire study. Per group 6-8 rats wereincluded. A vehicle group and positive control group were included ineach set of tests. Rats were dosed subcutaneously (sc) once in themorning 1 hour before turning off the lights, using a bodyweight-corrected dose (30 nmol/kg) of test peptide. Dosing volume was 2ml/kg. Food intake was recorded online using an automated food intakesystem (HM02, MBRose, Denmark) for 4 days or manually at 0 h for apredose and then 24, 48, 72 and 96 h post dosing. Body weight wasmeasured daily.

Statistical analyses were performed using GraphPad™ Prism version 7. Themeasured parameters were compared using two-way ANOVA followed byDunnett's multiple comparison tests. Differences were consideredstatistically significant at p<0.05.

Results

48 h after dosing, each of the tested compounds (except for compounds 30and 37) had given rise to a clear, statistically significant inhibitionof food intake (vehicle-corrected, in %). This reduction in food intakewas reflected by a decrease in body weight (vehicle-corrected, in %).Normal feeding behavior was subsequently resumed.

TABLE 5 Effect on food intake and body weight Food intake reduction Bodyweight in % at 48 h reduction in % at 48 h Compound No. Dose: 30 nmol/kgDose: 30 nmol/kg 41 75 11 35 72 12 18 58 10 39 76 9.2 40 83 7.3 37 244.3 30 <20 0 36 44 (at dose of 10 nmol/kg) 5.2 (at dose of 10 nmol/kg) 434 4.6 38 43 8.2 20 49 7.8 15 71 7.7 16 66 7.4 3 34 5.6

Example 8

Acute Fed Basal Glucose Changes in Diabetic ZDF Rats

Male Zucker Diabetic Fatty rat (ZDF-Lepr^(fa)/Crl) are obtained fromCharles River, US. Animals are acclimatised to experimental conditionsfor at least 14 days before the start of the study. From arrival andthroughout the study, the rats are housed in groups of 2 (n=2) in alight-, temperature- and humidity-controlled room. Animals have accessad libitum to food (KLIBA 2437, Provimi Kliba AG, Switzerland) and water(domestic quality tap water) during the entire study.

Rats are randomized based on blood glucose, HbA1c and body weight into adesignated test and vehicle group. The total number of animals per groupis 10 (n=10). Rats are dosed subcutaneously (sc) once in the morningusing a body weight-corrected dose (10 nmol/kg) of test peptide. Dosingvolume is 2 ml/kg.

Blood glucose levels in tail blood samples taken 24, 72, 96 and 168hours post dosing and blood glucose levels are determined using aglucometer (GlucoSmart Swing™; MSP Bodmann GmbH, Germany).

Statistical analyses are performed using GraphPad™ Prism version 7. Themeasured parameters are compared using two-way ANOVA followed by Sidakmultiple comparison tests. Differences are considered statisticallysignificant at p<0.05.

1. An amylin analogue which is a compound having the formula:R¹—Z—R² wherein R¹ is hydrogen, C₁₋₄ acyl, benzoyl or C₁₋₄ alkyl, or ahalf-life extending moiety M, wherein M is optionally linked to Z via alinker moiety L; R² is OH or NHR³, wherein R³ is hydrogen or C₁₋₃-alkyl;and Z is an amino acid sequence of formula I: (I) (SEQ ID NO: 3)X1-X2-X3-X4-X5-X6-X7-Ala-Thr-X10-Arg-Leu-Ala-X14-Phe-Leu-X17-Arg-X19-X20-Phe-Gly(Me)-Ala-Ile(Me)-X27-Ser-Ser-Thr-Glu-X32-Gly-Ser-X35-Thr-X37

wherein X1 is selected from the group consisting of Arg, Lys and Glu; X3is selected from the group consisting of Gly, Gln and Pro; X4 isselected from the group consisting of Thr and Glu; X5 is selected fromthe group consisting of Ala and Leu; X6 is selected from the groupconsisting of Thr and Ser; X10 is selected from the group consisting ofGlu and Gln; X14 is selected from the group consisting of Aad, His, Asp,Asn and Arg; X17 is selected from the group consisting of Gln, His andThr; X19-X20 is selected from Ser-Ser, Thr-Thr, Ala-Thr, Ala-Ala,Gly-Thr, Gly-Gly and Ala-Asn or is absent; X27 is selected from thegroup consisting of Leu and Pro; X32 is selected from the groupconsisting of Val and Thr; X35 is selected from the group consisting ofAsn and Ser; X37 is selected from the group consisting of Hyp and Pro;and X2 and X7 are amino acid residues whose side chains together form alactam bridge; or a pharmaceutically acceptable salt thereof.
 2. Anamylin analogue according to claim 1 wherein X1 is selected from Arg andLys.
 3. An amylin analogue according to claim 1 wherein: (i) X3 is Gly,X4 is Thr, X5 is Ala and/or X6 is Thr; (ii) X3 is Gly, X4 is Thr, X5 isAla and X6 is Thr; (iii) X14 is selected from His, Asp and Aad; (iv) X17is Gln; (v) X19-X20 is selected from Ser-Ser and Thr-Thr, or is absent;or (vi) X32 is Val, X35 is Asn and/or X37 is Hyp. 4-8. (canceled)
 9. Anamylin analogue according to claim 1 wherein Z is an amino acid sequenceof formula II: (II) (SEQ ID NO: 4) X1-X2-Gly-Thr-Ala-Thr-X7-Ala-Thr-X10-Arg-Leu-Ala-X14-Phe-Leu-Gln-Arg-X19-X20-Phe-Gly(Me)-Ala-Ile(Me)-X27-Ser-Ser-Thr-Glu-Val-Gly-Ser-Asn-Thr-Hyp

wherein X1 is selected from Arg and Lys; X10 is selected from the groupconsisting of Glu and Gln; X14 is selected from the group consisting ofAad, Asp and His; X19-X20 is selected from Ser-Ser and Thr-Thr or isabsent; X27 is selected from the group consisting of Leu and Pro; and X2and X7 are amino acid residues whose side chains together form a lactambridge.
 10. (canceled)
 11. An amylin analogue according to claim 1wherein one of the residues at position X2 and X7 is selected from Asp,Glu and Aad, and the other is selected from Dap, Dab, Orn, Lys and hLys.12. (canceled)
 13. An amylin analogue according to claim 11 wherein: X2is Asp and X7 is Lys; X2 is Asp and X7 is Orn; X2 is Asp and X7 is Dab;X2 is Asp and X7 is hLys; X2 is Dap and X7 is Aad; X2 is Glu and X7 isDab; or X2 is Dab and X7 is Glu.
 14. An amylin analogue according toclaim 13 wherein: X2 is Asp and X7 is Lys; or X2 is Asp and X7 is Orn.15. An amylin analogue according to claim 1 wherein X14 is selected fromAsp and Aad.
 16. An amylin analogue according to claim 1 wherein Z is anamino acid sequence selected from the group consisting of:(SEQ ID NO: 5)RD()GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 6)RD()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 7)RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 8)RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 9)RD()GTAT-Orn()-ATERLAHFLQRF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 10)RD()GTAT-Orn()-ATERLAHFLHRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNTP(SEQ ID NO: 11)RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 12)RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 13)RD()GTAT-Orn()-ATERLARFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 14)ED()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 15)RD()GEATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 16)RD()GTLTK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 17)RD()GTASK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 18)RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 19)RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 20)RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 21)RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 22)RD()GTAT-hLys()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 23)RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 24)RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 25)RD()GTAT-Orn()-ATERLA-Aad-FLTRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSST-Hyp(SEQ ID NO: 26)RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 27)RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 28)RD()GTAT-Orn()-ATERLA-Aad-FLQRATF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 29)RD()GTAT-Orn()-ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 30)RD()GTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 31)RD()QTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 32)RD()PTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 33)ED()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 34)RD()GTATK()ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 35)RD()GTATK()ATERLA-Aad-FLQRGGF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 36)RD()GTATK()ATERLA-Aad-FLQRANF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 37)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp(SEQ ID NO: 38)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 39)RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 40)ED()GTATK()ATERLA-Aad-FLQRSSFGly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 41)RD()GTATK()ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 42)KD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-AIle(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 43)RD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 44)RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 45)RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 46)KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 47)KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp(SEQ ID NO: 48)R-Dap()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 49)R-Dab()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 50)R-Orn()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 51)R-Dap()-GTAT-Aad()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 52)R-Dab()-GTATE()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 53)R-Aad()-GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp(SEQ ID NO: 54)RE()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp

or a pharmaceutically acceptable salt thereof.
 17. An amylin analogueaccording to claim 1 wherein R¹ is M or M-L-.
 18. An amylin analogueaccording to claim 17 wherein M is a lipophilic substituent comprising ahydrocarbon chain having from 10 to 24 C atoms.
 19. An amylin analogueaccording to claim 18 wherein the lipophilic substituent comprises acarboxylic acid group at the end of the hydrocarbon chain.
 20. An amylinanalogue according to claim 19 wherein the lipophilic substituent is a15-carboxy-pentadecanoyl, 17-carboxy-heptadecanoyl or19-carboxy-nonadecanoyl moiety.
 21. An amylin analogue according toclaim 17 wherein the linker L comprises a residue of Gly, Pro, Ala, Val,Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln, Asn, α-Glu,γ-Glu, ε-Lys, Asp, β-Asp, Ser, Thr, Gaba, Aib, β-Ala (i.e.,3-aminopropanoyl), 4-aminobutanoyl, 5-aminopentanoyl, 6-aminohexanoyl,7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl, 10-aminodecanoyl or8Ado (i.e., 8-amino-3,6-dioxaoctanoyl).
 22. An amylin analogue accordingto claim 21 wherein L is a residue of Glu, γ-Glu, ε-Lys, β-Ala,4-aminobutanoyl, 8-aminooctanoyl or 8Ado.
 23. An amylin analogueaccording to claim 22 wherein R¹ is [19CD]-isoGlu.
 24. An amylinanalogue according to claim 1 wherein R₂ is NH₂.
 25. An amylin analogueaccording to claim 1 which is: (Compound 1) (SEQ ID NO: 55)[19CD]-isoGlu-RD()GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 2) (SEQ ID NO: 56)[19CD]-isoGlu-RD()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 3) (SEQ ID NO: 57)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 4) (SEQ ID NO: 58)[19CD]-isoGlu-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 5) (SEQ ID NO: 59)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 6) (SEQ ID NO: 60)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLQRF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 7) (SEQ ID NO: 61)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLAHFLHRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNTP-NH₂ (Compound 8) (SEQ ID NO: 62)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 9) (SEQ ID NO: 63)[19CD]-isoGlu-RD()GTAT-Orn()-ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 10) (SEQ ID NO: 64)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLARFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 11) (SEQ ID NO: 65)[19CD]-isoGlu-ED()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 12) (SEQ ID NO: 66)[19CD]-isoGlu-RD()GEATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 13) (SEQ ID NO: 67)[19CD]-isoGlu-RD()GTLTK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 14) (SEQ ID NO: 68)[19CD]-isoGlu-RD()GTASK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 15) (SEQ ID NO: 69)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 16) (SEQ ID NO: 70)[19CD]-isoGlu-RD()GTATK()ATQRLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 17) (SEQ ID NO: 71)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-PSSTE-VGSNT-Hyp-NH₂ (Compound 18) (SEQ ID NO: 72)[19CD]-isoGlu-RD()GTATK()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 19) (SEQ ID NO: 73)[19CD]-isoGlu-RD()GTAT-hLys()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 20) (SEQ ID NO: 74)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 21) (SEQ ID NO: 75)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 22) (SEQ ID NO: 76)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLTRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSST-Hyp-NH₂ (Compound 23) (SEQ ID NO: 77)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 24) (SEQ ID NO: 78)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRTTF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 25) (SEQ ID NO: 79)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRATF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 26) (SEQ ID NO: 80)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 27) (SEQ ID NO: 81)[19CD]-isoGlu-RD()GTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 28) (SEQ ID NO: 82)[19CD]-isoGlu-RD()QTAT-Orn()-ATERLA-Aad-FLQRGTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 29) (SEQ ID NO: 83)[19CD]-isoGlu-RD()PTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 30) (SEQ ID NO: 84)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 31) (SEQ ID NO: 85)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRAAF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 32) (SEQ ID NO: 86)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRGGF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 33) (SEQ ID NO: 87)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRANF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 34) (SEQ ID NO: 88)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-PSSTEVGSNT-Hyp-NH₂ (Compound 35) (SEQ ID NO: 89)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 36) (SEQ ID NO: 90)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 37) (SEQ ID NO: 91)[19CD]-isoGlu-ED()GTATK()ATERLA-Aad-FLQRSSFGly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 38) (SEQ ID NO: 92)[19CD]-isoGlu-RD()GTATK()ATERLA-Aad-FLORTTF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 39) (SEQ ID NO: 93)[19CD]-isoGlu-KD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-AIle(Me)-LSSTEVGSNTHyp-NH₂ (Compound 40) (SEQ ID NO: 94)[19CD]-isoGlu-RD()GTATK()ATQRLA-Aad-FLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 41) (SEQ ID NO: 95)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 42) (SEQ ID NO: 96)[19CD]-isoGlu-RD()GTATK()ATQRLADFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 43) (SEQ ID NO: 97)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTE-VGSNT-Hyp-NH₂ (Compound 44) (SEQ ID NO: 98)[19CD]-isoGlu-KD()GTATK()ATQRLANFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTETGSNT-Hyp-NH₂ (Compound 45) (SEQ ID NO: 99)[19CD]-isoGlu-R-Dap()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 46) (SEQ ID NO: 100)[19CD]-isoGlu-R-Dab()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 47) (SEQ ID NO: 101)[19CD]-isoGlu-R-Orn()-GTATD()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 48) (SEQ ID NO: 102)[19CD]-isoGlu-R-Dap()-GTAT-Aad()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 49) (SEQ ID NO: 103)[19CD]-isoGlu-R-Dab()-GTATE()ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 50) (SEQ ID NO: 104)[19CD]-isoGlu-R-Aad()-GTAT-Dap()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂ (Compound 51) (SEQ ID NO: 105)[19CD]-isoGlu-RE()GTAT-Dab()-ATERLAHFLQRSSF-Gly(Me)-A-Ile(Me)-LSSTEVGSNT-Hyp-NH₂

or a pharmaceutically acceptable salt thereof.
 26. A pharmaceuticalcomposition comprising an amylin analogue according to claim 1 incombination with a pharmaceutically acceptable carrier, excipient orvehicle.
 27. A method for the synthesis of an amylin analogue accordingto claim 1 comprising synthesizing the analogue by solid-phase orliquid-phase peptide synthesis methodology, optionally isolating and/orpurifying the final product, and optionally further comprising the stepof forming an amide bond between the side chains at positions 2 and 7.28-32. (canceled)